The Mona Lisa at the Prado in Madrid was thought to be just another fine copy, with added eyebrows and an odd black background. But curators at Spain's national art museum yesterday announced a startling discovery: the painting was actually executed by an artist in Leonardo da Vinci's workshop at the same time as the original.

It is the first known copy of the most famous painting in history, and a discovery that curators believe sheds new light on the creation of the masterpiece.

Deputy conservator, Gabriele Finaldi, said: "It's as if we were standing in the workshop itself, and at the next easel. You can see that the artist was working step by step with Leonardo. When Leonardo made a change, he made a change."

The copy sits in a dimly-lit room awaiting the finishing touches of a two-year restoration, during which its true origin was revealed. Curators decided it needed a face lift because it was going on loan to the Louvre in March. Following X-ray and infrared studies, they were surprised to find a landscape hidden beneath the dark paint behind the subject.

Conservators believe the artist could be Francesco Melzi, one of Leonardo's favourite pupils. "When you look at the copy, you can imagine that this is what the Mona Lisa looked like in the 16th century," Mr Finaldi continued. "It's not just the details and the colour use. It has also been protected from light and dirt for centuries. So what you see if a very reliable appearance."

The Mona Lisa is widely believed to be a portrait of Lisa Gherardini, a Florentine merchant's wife, and the copy makes her look younger and more seductive.

Miguel Falomir, chief conservator for renaissance painting, said: "When the X-ray revealed the landscape, we saw it was in absolutely extraordinary condition. It was the most surprising thing to emerge in the conservation workshop in the 14 years I've been at the Prado."

The copy has belonged to the museum at least since the 1666, first as part of the royal collection and then as a state treasure. It was first thought to have been produced by a Flemish hand after da Vinci's death. Then it was believed to be a later Italian copy.

The Art Newspaper, which first published the remarkable discovery, said: "This sensational picture will transform our understanding of the world's most famous picture."

The "Prado Mona Lisa" was beloved by many visitors, even if that odd black background didn't highlight La Gioconda's beauty as does the landscape. "For 400 years, it has had the same owner, the same atmospheric conditions," said Mr Falomir. "It has never left the Prado. It's probably in better shape than the original."

Secret masterpieces: Art undercover

Painted by an unknown artist and inauspiciously named simply 'Old Man with a Beard', a portrait dating from the 1630s was only identified as a lost work by Rembrandt after it was scanned by X-rays last year. The technology revealed another image beneath the surface that had been painted over – which turned out to be a self-portrait by the Dutch Master.

'Portrait Of Don Ramó* Satué' had long been recognised as a masterpiece by Francisco de Goya, but scans last year revealed that he had previously used the same canvas for an unfinished painting of a French general. With the mystery figure thought to be Napoleon Bonaparte's brother, Joseph, who served as King of Spain between 1808 and 1813, the original picture may have been painted over for political reasons following the end of his reign.

A woman's head was revealed to lie beneath Vincent Van Gogh's 'Patch of Grass' by X-rays, before an advance in equipment in 2008 allowed scientists to show to a remarkable degree the brown hues of the older image under the greens and yellows of the new picture.