Scientists say they have confirmed the theory that Australian racehorse legend Phar Lap was killed by arsenic poisoning.

For years argument has raged over who and what killed Phar Lap, the horse that won the 1930 Melbourne Cup and for a time dominated Australia's horse racing scene.

But a fortnight after winning North America's richest race, the Agua Caliente Handicap, in 1932, the horse was struck down by a mystery illness.

Some believed the horse was poisoned by gangsters, while others argued he died accidentally after consuming insecticide or from acute bacterial gastroenteritis.

Associate Professor Ivan Kempson, from Taiwan's Institute of Physics, Academia Sinica, and Dermot Henry from Museum Victoria have been investigating the death.

"All our observations are consistent with consumption of a large dose of arsenic," Dr Kempson said.

"It definitely re-highlights the arsenic theory of death because it's been quite debatable."

Dr Kempson and Mr Henry say they now have the best evidence to date that backs earlier claims arsenic is to blame.

Dr Kempson said arsenic poisoning was initially ruled out after Phar Lap's autopsy. But he and Mr Henry have been the first to examine samples of hair, which is well known for storing toxic elements.

Dr Kempson used the Advanced Photon Source synchrotron near Chicago to analyse samples of hair taken from the back of Phar Lap's neck.

The analysis of the hair, taken from the stuffed horse at Museum Victoria in Melbourne, was able to detect the chemical structure of arsenic.

The researchers also studied the distribution of the arsenic in cross sections of the hair and along its length.

Earlier criticisms of the arsenic theory suggested the arsenic detected in the hair could have come from other sources such as the taxidermy process itself.

Taxidermists used to paint arsenic on the skin of dead animals to preserve it.

To determine whether the arsenic came from taxidermy or from poisoning, Dr Kempson and Mr Henry compared the arsenic found in Phar Lap's hair with that from other museum specimens which had been preserved using arsenic.

They analysed samples of hair from the back of the neck of early 20th century animals preserved at the Museum of Scotland.

The two horses, a pony and a zebra were treated with arsenic during taxidermy but presumed not to have been poisoned by arsenic.

Dr Kempson says hair from these animals showed very different arsenic chemistry and distribution to the hair from Phar Lap.

He says when arsenic is absorbed into the hair from the outside, for examply from taxidermy, its chemical form is very different to that when it is ingested.

"If you think of carbon, for example, carbon that is in a graphite pencil is very different to carbon that is in a diamond," Dr Kempson said.

"Even though it's all carbon it is very different, based on the chemistry. It's the same for the arsenic as well."

Dr Kempson says while hair from the Scottish animals showed arsenic in the absorbed form, Phar Lap's hair contained mainly arsenic in the ingested form.

Studies of arsenic distribution in the hair also revealed stark differences between Phar Lap and the Scottish animals.

Cross sections of the hairs showed arsenic in Phar Lap was mainly bound to sulphur in the hair, whereas in the Scottish animals it was mostly bound to melanin.

Dr Kempson said examination of arsenic distribution along the length of the hair also showed a massive peak just below the skin level in Phar Lap's hair, consistent with poisoning from heavy metals such as mercury and lead.

In the Scottish animals arsenic was evenly distributed along the hair length, consistent with having been applied as a solution over the whole animal hide.

"This is really the only physical evidence that exists on the cause of death and it is consistent with poisoning," Dr Kempson said.

As to who it was who might have poisoned Phar Lap, Dr Kempson says we will probably never know.

"All we've shown is that he consumed arsenic," he said.

Dr Kempson and Mr Henry report their findings online ahead of print publication in Angewandte Chemie International Edition.