Last year's pole man Andrea Iannone utilised the searing top speed of the Ducati to snatch the top spot in FP2 by almost half-a-second at Mugello.
After a damp morning session, dry conditions prevailed in the afternoon and Iannone ended the day in P1 on the Desmosedici, toppling Jorge Lorenzo.
Many of the riders, including Lorenzo, were out on track for the first time on Friday after opting to sit out FP1.
Iannone – bound for Suzuki in 2017 – powered through with a time of 1m 47.696s to head MotoGP World Championship leader Lorenzo on the Movistar Yamaha by 0.461s, with Ducati test rider Michele Pirro third quickest, finishing 0.549s off the leader after benefiting from a tow from Lorenzo.
Aleix Espargaro slotted into fourth on the Ecstar Suzuki with a lap in 1m 48.270s while the top six was rounded out by early pacesetter and Marc Marquez on the Repsol Honda and home favourite Valentino Rossi.
Rossi, who used FP1 to practice his race starts out of pit lane, was six tenths behind his Movistar Yamaha team-mate Lorenzo, clocking his best lap in 1m 48.340s.
Young Spaniard Maverick Vinales, who will join Rossi at Yamaha next season, was seventh quickest on the Suzuki GSX-RR ahead of Britain's Scott Redding, who led briefly on the Pramac Ducati in the final moments before being shunted back to eighth.
Cal Crutchlow (LCR Honda) and Pol Espargaro on the Monster Yamaha Tech 3 M1 completed the top ten, seven tenths off Iannone's pace.
Dani Pedrosa, who this week confirmed a new two-year deal to stay at Repsol Honda after rumours linking him to Yamaha, was down in 11th place, one second adrift of first position and narrowly ahead of Danilo Petrucci (Pramac Ducati).
It was a disappointing afternoon also for Andrea Dovizioso who, in contrast to team-mate Iannone, was some way out of contention in 13th with 1.1 seconds to find.
FP1 pacesetter Yonny Hernandez (Aspar Ducati) ended the day in 14th ahead of Bradley Smith, who also has work to do on the Monster Yamaha Tech 3.
Eugene Laverty came off in the session on the second of the Aspar Ducati machines with around 18 minutes remaining, finishing 19th on the time sheets.
Loris Baz, who had a problem with his number one bike, was forced to park it up and return to pit lane for his second Avintia Ducati. Baz finished 22nd behind Aprilia pair Alvaro Bautista and Stefan Bradl.
His team-mate, Hector Barbera, was 16th ahead of Marc VDS Honda riders Tito Rabat and Jack Miller.
1. Andrea Iannone
2. Jorge Lorenzo
3. Michele Pirro
4. Aleix Espargaro
5. Marc Marquez
6. Valentino Rossi
7. Maverick Viñales
8. Scott Redding
9. Cal Crutchlow
10. Pol Espargaro
11. Dani Pedrosa
12. Danilo Petrucci
13. Andrea Dovizioso
14. Yonny Hernandez
15. Bradley Smith
16. Hector Barbera
17. Tito Rabat
18. Jack Miller
19. Eugene Laverty
20. Alvaro Bautista
21. Stefan Bradl
22. Loris Baz