For its home race, Aprilia has five RS-GPs on the grid with its two factory team riders, the Trackhouse Racing duo and wildcard Lorenzo Savadori.
Vinales was the best of them at the top of the timesheets in the first 45-minute practice of the Mugello weekend, the Spaniard setting a 1m46.140s on a fresh medium rear tyre in the closing stages.
He headed 2021 world champion Quartararo on the factory Yamaha, with Franco Morbidelli leading the Ducati charge on his Pramac-run GP24.
Marc Marquez set the early pace on his Gresini Ducati as he puts the rider market talk on his future to one side, the six-time MotoGP world champion posting a 1m47.002s.
Yamaha’s Alex Rins then took over moments later with a 1m46.719s, before Marquez returned to the top of the order with half an hour remaining.
Marquez’s 1m46.587s stood as the benchmark for the next 20 minutes before Tech3 GasGas rookie Pedro Acosta – fitted with a new medium rear tyre – fired in a 1m46.459s.
Vinales edged ahead with a 1m46.140s five minutes later, which would cement him top spot as the chequered flag came out.
Quartararo put in a late 1m46.328s to jump up to second, with Yamaha having tested recently at Mugello.
Morbidelli completed the top three from Acosta, who crashed late on at Turn 10 while pushing for a session-best lap.
Championship leader Jorge Martin – who looks set to join the factory Ducati squad next year – completed the top five having been one of only two riders alongside team-mate Morbidelli to fit a fresh soft tyre in FP1.
Marc Marquez was sixth having stuck with the same tyres all session, while Aprilia’s Aleix Espargaro was seventh ahead of Rins, who suffered an early crash at Turn 1.
Francesco Bagnaia looks to extend his run of Italian GP wins to three in succession this weekend on his factory team Ducati and was ninth in FP1, with Alex Marquez 10th on the second Gresini Ducati.
The top factory team KTM was Jack Miller in 12th, while Pol Espargaro was 15th as he makes a wildcard appearance for the Austrian manufacturer at Mugello.
Luca Marini was the top Honda in 19th as the factory team HRC riders run the new aero Johann Zarco raced with last weekend at Barcelona.
Savadori was last on the fifth Aprilia.