The Ducati rider had led the field during the first half of the session as the weather conditions restricted riders from dipping below the 2m23s bracket.
But as the light rain eased and eventually stopped in the final 10 minutes, times improved by around four seconds.
That allowed Rossi to knock his team-mate Nicky Hayden off the top spot by 0.077 seconds with a time of 2m19.328s.
Andrea Dovizioso was another to set a late flyer, the Tech 3 Yamaha rider snatching third after the chequered flag and knocking the Honda of world champion Casey Stoner back to fourth.
Dovizioso's British team-mate Cal Crutchlow briefly held the top spot with eight minutes remaining, but fell back to fifth at the end.
He was followed by Stefan Bradl's LCR Honda, Dani Pedrosa's factory example and the Yamaha of world championship leader Jorge Lorenzo.
Gresini Honda rider Alvaro Bautista was ninth, despite crashing at Stowe with nine minutes remaining - just a minute after Ben Spies high-sided his Yamaha at the same place.
Hector Barbera completed the top 10 on his Pramac Ducati with Randy de Puniet the best of the CRT riders in 12th on his Aspar Aprilia.
Only 11 MotoGP prototypes are in the field, as Cardion Ducati's Karel Abraham has been forced to sit out the event due to a hand injury from testing.
Opening practice standings:
1. Valentino Rossi
2. Nicky Hayden
3. Andrea Dovizioso
4. Casey Stoner
5. Cal Crutchlow
6. Stefan Bradl
7. Dani Pedrosa
8. Jorge Lorenzo
9. Alvaro Bautista
10. Hector Barbera
11. Ben Spies
12. Randy De Puniet
13. Mattia Pasini
14. Danilo Petrucci
15. Michele Pirro
16. Aleix Espargaro
17. James Ellison
18. Yonny Hernandez
19. Ivan Silva
20. Colin Edwards
