Casey Stoner threw down the gauntlet to the Yamaha duo by setting the pace in the final free practice session before qualifying at Mugello.
The Ducati rider lapped in 1m49.323s on only his fifth lap of the morning, beating Jorge Lorenzo's Friday-topping time - which was itself under the lap record - by 0.2 seconds.
Stoner's mark remained unbeatable for the rest of the hour, although Lorenzo eventually closed to within 0.027s of the Ducati. Valentino Rossi, who is chasing an eighth straight Mugello win, was third.
Toni Elias jumped to a surprise fourth for Gresini Honda with his final lap, pushing Suzuki's Loris Capirossi back to fifth, ahead of Colin Edwards (Tech 3 Yamaha) and the two factory Hondas.
While one works Ducati led the way, the other propped up the order, with Nicky Hayden slowest of all as his troubled season failed to improve on the team's home ground.
But there was encouragement for another struggling Ducati rider, as Pramac's Niccolo Canepa showed how much his lack of circuit knowledge has previously held him back by taking 12th. The Italian rookie has been mired at the tail of the field for most of 2009 so far, but knows Mugello well from his days as a Ducati test rider.
Pos Rider Bike Time
1. Casey Stoner Ducati 1m49.323s
2. Jorge Lorenzo Yamaha 1m49.350s + 0.027s
3. Valentino Rossi Yamaha 1m49.517s + 0.194s
4. Toni Elias Gresini Honda 1m49.954s + 0.631s
5. Loris Capirossi Suzuki 1m49.981s + 0.658s
6. Colin Edwards Tech 3 Yamaha 1m50.034s + 0.711s
7. Andrea Dovizioso Honda 1m50.036s + 0.713s
8. Dani Pedrosa Honda 1m50.154s + 0.831s
9. Alex de Angelis Gresini Honda 1m50.364s + 1.041s
10. Randy de Puniet LCR Honda 1m50.617s + 1.294s
11. James Toseland Tech 3 Yamaha 1m51.017s + 1.694s
12. Niccolo Canepa Pramac Ducati 1m51.313s + 1.990s
13. Chris Vermeulen Suzuki 1m51.342s + 2.019s
14. Yuki Takahashi Scot Honda 1m51.392s + 2.069s
15. Marco Melandri Hayate Kawasaki 1m51.400s + 2.077s
16. Mika Kallio Pramac Ducati 1m51.723s + 2.400s
17. Nicky Hayden Ducati 1m52.108s + 2.785s