Madrid's Prado Museum unveiled a restored version of the so-called "Mona Lisa of the Prado", in which the woman the Italian artist painted, with her enigmatic smile, sat previously against a background of black paint.
The restored version, with the black paint removed, shows her backed by a landscape of hills and rivers resembling those of the original masterpiece, which hangs in the Louvre museum in Paris.
Previously thought to be a later replica, the Prado's picture is in fact "probably the first known copy of La Joconda", as the Mona Lisa is known, said Miguel Falomir, a curator of Italian painting at the Prado.
He said expert analysis suggests "the painting was done in the painter's own workshop. It is absolutely consistent with Leonardo's work".
But he added: "It is a work in which Leonardo himself did not intervene."
The museum said the painting will go on show at the Louvre alongside the original from March 26.
The experts said that techniques used in the Prado's painting, now brought to light through methods such as infrared scanning, shed precious light on the way Leonardo himself worked.
"The picture was well-known but we know it much better now. We did not know what lay underneath" the black paint, which was added in the 18th century for unknown reasons, said Gabriele Finaldi, a deputy director of the Prado.
"This is very, very close to how the painting looked in 1505" when Leonardo finished his masterpiece, he said.
As well as the landscape, the restoration has revealed earlier elements that were painted over, shedding light on how both portraits were likely composed.
"Our colleagues at the Louvre now have a whole lot more information they can use in their research on their own painting," Finaldi said.
Art experts believe the woman in the painting to be Lisa Gherardini, the wife of a Florentine cloth merchant named Francesco del Giocondo.
In the Prado's painting, unveiled to journalists in the museum's vaults on Wednesday, she appears younger and fresher-faced than the one in Paris, and the landscape of rivers and hills lusher.
Finaldi said this was because the original Mona Lisa is "very dirty".
"When paintings are dirty, the sitters tend to look older," he told AFP.
The specialist British journal The Art Newspaper, which first reported the results of the painting's restoration, hailed the new-look Mona Lisa and the discovery of what lay under the black paint.
"This sensational find will transform our understanding of the world's most famous picture," it wrote.