A museum spokeswoman said the copy was done alongside the 16th-century original, which now hangs in the Louvre Museum in Paris, apparently making it the earliest replica ever found.
The Prado said it didn't realize the significance of its copy of the "Mona Lisa" until a restoration revealed hidden layers. There are dozens of the surviving replicas of the masterpiece from the 16th and 17th centuries.
The Madrid version shows the same woman, but prior to the restoration it lacked the landscape background and was covered with paint and varnish. The spokeswoman said the painting had once been on display in the museum but had always been considered a poor copy.
Museum director Miguel Zugaza presented the work at a news conference later Wednesday.
El Pais newspaper said the Louvre supports the Prado's new evaluation of the painting. Calling the painting 'Mona Lisa's twin,' El Mundo cited museum officials as saying the Prado copy was better preserved in several areas than the original and would help with studies of the masterpiece.
The replica was restored as part of plans for it to be included in a Louvre exhibition on Leonardo later this year. The Prado plans to put it on display later this month before it travels to France.
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