Researchers who drilled through a fresco in Florence's town hall say paint used by Leonardo was found in the cavity behind

Researchers in Florence have claimed they have proof that a lost masterpiece by Leonardo da Vinci, entitled The Battle of Anghiari, exists on a wall in a cavity in Florence's town hall, where it has been hidden for five centuries.

Late last year researchers drilled tiny holes in a later fresco on the wall in the Palazzo Vecchio which conceals the cavity. They inserted a 4mm wide probe to film inside and retrieved samples of paint. They said the paint was similar to that used by Leonardo for the Mona Lisa.

"We need the courage to push on and resolve this mystery," said Matteo Renzi, the mayor of Florence, who is urging the Italian government to approve removal of parts of the later fresco, Giorgio Vasari's The Battle of Marciano in Val di Chiana, which was painted in 1563.

Leonardo, working in the Hall of Five Hundred in the Palazzo Vecchio in 1504, completed only the centrepiece of his work. This was later copied by Rubens, whose drawing hangs in the Louvre. After 1555 the palace room was renovated and Leonardo's half-finished painting was believed lost forever.

A team led by Maurizio Seracini, a researcher at the University of California, San Diego, obtained permission last year to drill six holes through Vasari's fresco in search of Leonardo's work.

A sample is taken from the Vasari wall. Dave Yoder/AFP/Getty

The team found the 3cm-4cm (less than 2in) cavity, previously spotted with radar, behind masonry. "No other gaps exist behind the other five massive Vasari frescoes in the high-ceilinged hall," the team said.

A sample of black material removed from the back wall was analysed with a scanning electron microscope and was found to be similar to black pigment found by the Louvre in brown glazes on the Mona Lisa and the painting St John the Baptist, the team said.

"Leonardo painted the Mona Lisa in Florence at the same time," said Seracini. "It appears to be a pigment used by [him] and not by other artists."

Flakes of red material were identified as being possibly lacquer. "This type of material is unlikely to be present in an ordinary plastered wall," the team said.

High-definition endoscopic images also revealed beige material on the original wall that "could only have been applied by a paint brush".

Some art experts have petitioned against Seracini drilling through the Vasari fresco, claiming any paint found behind might have been left by another artist.

Speaking at a conference held in front of the shrouded wall on Monday, the head of Florence's state restoration centre, Marco Ciatti, said he was not yet convinced the Leonardo work was there.

Seracini said the holes had been drilled only in peripheral areas of the fresco which had been restored: he would now be asking permission to drill in other restored areas, within 10 sq metres at the centre of the Vasari fresco.

Seracini's suspicions that Vasari did not want to destroy Leonardo's work, preferring to add his own fresco over it, were reinforced in the 1970s, he said, when he found the artist had painted a soldier in his fresco with a flag upon which was written: "He who seeks, finds."

Seracini said a 10-sq-metre section of another giant Vasari fresco in the room – depicting an injured horse lying beside a bloodied warrior pulling a spear out of his back – had been removed and then replaced in a search for the Battle of Anghiari in the 1980s.