Ducati's Sete Gibernau set the pace in last year's Barcelona race only to see Rossi take victory.
BBC: SPORT | Motorsport | Motorbikes | Rossi targets Barcelona hat-trick
Rossi hit back straight away to cruise into an early lead, and Gibernau's exit boosted his victory hopes.
BBC: SPORT | Motorsport | Motorbikes | Rossi holds off Biaggi at Mugello
Sete Gibernau is already out of this week's race after crashing last weekend.
BBC: SPORT | Motorsport | Motorbikes | Rossi aims to race despite crash
Gibernau had been seeking revenge over the world champion for the last-lap incident which cost him a win at Jerez.
BBC: SPORT | Motorsport | Motorbikes | Rossi snatches win from Gibernau
Rossi was lucky to avoid the horror crash which marred the start of last Sunday's Catalunya Grand Prix, in which Gibernau broke his collarbone.
Rossi's chief rival for honours this season has been Sete Gibernau and the Movistar Honda rider expects to be a factor once again at Mugello.
BBC: Rossi leads the MotoGP standings by 37 points
Gibernau had lost out to Rossi in more dramatic fashion in the first race of the season at Jerez after their bikes came together on the final lap.
Honda rider Gibernau led for most of the 25 laps in Barcelona, only for Rossi to pass him on lap 23 to take his fifth win of the season on the Yamaha.
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