The following correction was printed in the Guardian's Corrections and clarifications column, Monday March 3 2008

In the report below (regarding new interpretations of forensic evidence relating to the assassination of Robert Kennedy) we stated that he was hit by four shots. However, only three bullets entered his body; the fourth was lodged in a shoulder pad. One of the pistols referred to was a Harrington & Richardson and not, as we had it, a Harrison & Richardson. This has been corrected.

The official record states that senator Robert F Kennedy, like his brother before him, was killed by a crazed lone gunman. But the assassination of a man who seemed to embody so much hope for a bitterly divided country embroiled in an unpopular war still troubles this nation.

Little about the official explanation of the events at the Ambassador Hotel on June 5 1968 makes sense. Now a new forensic analysis of the only audio recording of the fatal shots has given new weight to a controversial theory that there were in fact two shooters, and that the man convicted of Kennedy's killing — Sirhan Sirhan - did not fire the fatal shots.

Following his victory speech to supporters after clinching a tight democratic primary victory in California, Kennedy left the podium in the Embassy ballroom to address a press conference.

Bloody mayhem

But the shortcut he and his entourage took through the hotel's pantry quickly descended into bloody mayhem. As Kennedy turned from shaking hands with two of the kitchen staff, a gunman stepped forward and began firing. Kennedy was hit by four shots including one which lodged in the vertebrae in his neck and another which entered his brain from below his right ear. He died in hospital the following day. Five other people were injured but survived.

Sirhan - a Palestinian refugee who said he wanted to "sacrifice" Kennedy "for the cause of the poor exploited people" - was quickly apprehended. He was eventually sentenced to life imprisonment.

"Sirhan was apprehended at the scene with literally a smoking gun," said acoustic forensic expert Philip Van Praag of PVP Designs, who has carried out the new analysis. "At the beginning many people looked upon this as an open-and-shut case. It was one man, Sirhan Sirhan, who was observed by a number of people, who aimed and fired a gun in the direction of Kennedy's entourage."

Close range

But the lone gunman explanation has always looked shaky. The autopsy of Kennedy's body suggested that all four shots that hit him came from behind, and powder marks on his skin showed they must have been from close range.

But Sirhan was in front of Kennedy when he fired, and after shooting two shots was overcome by hotel staff, who pinned him to a table. Also, Sirhan fired eight shots in total, yet 14 were found lodged around the room and in the victims.

"There is no doubt in our minds that no fewer than 14 shots were fired in the pantry on that evening and that Sirhan did not in fact kill Senator Kennedy," said Robert Joling, a forensic scientist who has been involved with the Kennedy case for nearly 40 years. He and Van Praag have published a book on the killing this week entitled "An Open and Shut Case".

The inconsistencies in the case have bred numerous conspiracy theories, including the involvement of the CIA and the idea that Sirhan - who claims not to remember the shooting and pleaded insanity at his trial - was a "Manchurian Candidate" assassin who was hypnotically programmed to kill the senator.

Audio recording

Now Van Praag has added new weight to the 'two shooters' theory. He reanalysed the only audio recording of the shooting, which was made by an independent journalist, Stanislaw Pruszynski. "At the time Pruszynski was not even aware that his recorder was still on," said Van Praag.

The recording quality is poor, but it is possible to make out 13 shots over the course of just over 5 seconds, before what Van Praag describes as "blood-curdling screams" obscure the sound. That is more than the eight rounds that Sirhan's cheap Iver Johnson Cadet 55 revolver carried.

Also, there are two pairs of double shots that occurred so close together it is inconceivable that Sirhan could have fired them all. The third and fourth shots and the seventh and eighth were separated by 122 and 149 milliseconds respectively. In tests, a trained firearms expert firing under ideal conditions could only manage 366 milliseconds between shots using the same weapon. And he was not being pinned to a table at the time.

Frequency anomalies

Lastly, five of the shots - 3, 5, 8, 10 and 12 in the sequence - were found to have odd acoustic characteristics when specific frequencies were analysed separately. Van Praag thinks this is because they came from a different gun pointing away from Pruszynski's microphone.

To recreate this he recorded the sounds made by firing the Iver Johnson and another revolver, a Harrington and Richardson 922. At least one member of Kennedy's entourage was carrying this weapon when the killing happened. In the acoustic tests it produced the same frequency anomalies Van Praag had seen in the original recording but only when fired away from the microphone.

He presented his results on Thursday at the American Academy of Forensic Sciences annual meeting in Washington DC.

Paul Schrade, a close associate of Kennedy's who was director of the United Auto Workers union, was at the senator's side in the pantry and was shot in the head. He told the meeting that America lost an outstanding leader and potentially great president that day.

Lost hope

"I think we were in a position of really changing this country," he said. "What we lost was a real hope and possibility of having a better country and having better relations around the world."

He wants to see the case reopened and properly investigated. "We're going to go ahead and do our best to find out who the second gunman was and that's going to take a lot of work," he said.

Van Praag also wants the case reexamined. "We would hope that the evidence that we have uncovered ... would make a strong enough case to get serious consideration once again by the authorities," he said.