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In 1506, Ezio Auditore, Niccolò Machiavelli, and Leonardo da Vinci visited the Lone Wolf in hopes of tracking down Micheletto Corella, who had been working on a plan to free Cesare Borgia from captivity.
After a brawl in the inn with Micheletto and his men, which resulted in Micheletto fleeing, Ezio, Niccolò and Leonardo stayed at the Lone Wolf themselves. When Cesare was subsequently freed from imprisonment by Micheletto, the inn acted as Cesare's main headquarters as he rebuilt his army.
However, after Ezio and Niccolò destroyed Cesare warships and military encampment with Leonardo's hand-held bombs, the two of them returned to spy on Cesare from the inn's rooftops. There, they witnessed him murder Micheletto, after he in turn had tried to kill Cesare.
Cesare eventually fled the inn and Valencia to cross the borders to the Navarre.