Behind the enigmatic smile: National Portrait Gallery to display 16th century work which has given up its secret after 400 years

The queen wears a magnificent gown and a faint, enigmatic smile ‑ but then she knows what she really holds in her hands, a secret revealed again to the world after more than 400 years.

Many portraits of Elizabeth I show her holding a posy, a conventional symbol of virginity or virtue. The National Portrait Gallery has discovered that in this portrait Gloriana originally held a far more disturbing object ‑ a serpent twined around her fingers.

A serpent can sometimes represent wisdom and judgment, as in the serpent and staff symbol of medicine, but in Christian iconography it is more often a symbol of sin or even the devil.

The unknown artist, painting around the late 1580s, clearly had a last-minute panic about the ambiguity of the image: the scaly blue-green and black serpent was painted out, and replaced with the safe ‑ if slightly oddly shaped ‑ posy.

Tarnya Cooper, curator of 16th century paintings at the gallery, who has led the research into several Tudor portraits about to be redisplayed, says the serpent is a unique attribute in portraits of the queen. "The portrait of Elizabeth I with a hidden serpent is a really unusual survival. Yet it is difficult to know exactly why the serpent may have been originally included, or how common this motif might have been. The queen certainly owned jewellery and costume including emblems of serpents, which were probably understood as a symbol of wisdom. However, no other portrait of Elizabeth appears to depict her holding a snake."

The gallery has owned the portrait for a century, but its condition is poor and it has not been on display since 1921. It will now be included in an exhibition opening later this month, Concealed and Revealed: The Changing Faces of Elizabeth I, of paintings made from the 1560s until just after the queen's death in 1603, which have all been altered in some way.

The x-rays that drove the serpent out of its lair also revealed another secret: the queen's exceptionally bumpy forehead is because of the inner woman trying to get out. The portrait was painted over an earlier, unfinished painting of another woman, probably by a different artist: the eyes and nose of the lost woman can just be seen in the queen's forehead.

Cooper said: "The recent technical analysis on these remarkable portraits has been critical to our understanding of Tudor painting."

The artist may never have seen the queen in the flesh ‑ and certainly, the art historians believe, never saw the serpent.

Concealed and Revealed, National Portrait Gallery London, 13 March