Jorge Lorenzo was fastest again in the third MotoGP practice session at Mugello but Marc Marquez will have to take part in the first qualifying session.
Lorenzo was in a class of his own for the opening half hour, improving his Friday benchmark early on before whittling it down to 1m47.972s aboard his Yamaha. He was the only rider below the 1m48s mark.
While the reigning champion progressed serenely through to Q2, Marquez will have to test his injured shoulder even more after missing the cut.
The Honda rider suffered another crash, though this was a low-speed tumble in the Turn 12 gravel, and he would go on to complete the session.
However, he could only manage the 12th fastest time and will be joined in Q1 by Andrea Iannone's Pramac Ducati - sixth after Friday - and Bradley Smith's Tech 3 Yamaha, with top CRT rider Aleix Espargaro delivering a superb lap to earn the final spot in Q2 on his Aspar Aprilia.
As Lorenzo remained unchallenged at the top of the timesheets, behind the places swapped almost every minute. First Andrea Dovizioso went second fastest before Dani Pedrosa and Stefan Bradl took over.
Eventually, it was Pedrosa's Honda which emerged as Lorenzo's closest challenger - significantly closer than on Friday, but still a quarter of a second behind.
Valentino Rossi and Cal Crutchlow were fourth and sixth respectively after low-key sessions ended with late improvements, while Nicky Hayden and wildcard Michele Pirro made it three Ducatis in the top 10.
Pos Rider Team/Bike Time Gap
1. Jorge Lorenzo Yamaha 1m47.972s
2. Dani Pedrosa Honda 1m48.222s + 0.250s
3. Stefan Bradl LCR Honda 1m48.489s + 0.517s
4. Valentino Rossi Yamaha 1m48.580s + 0.608s
5. Alvaro Bautista Gresini Honda 1m48.703s + 0.731s
6. Cal Crutchlow Tech 3 Yamaha 1m48.749s + 0.777s
7. Andrea Dovizioso Ducati 1m48.779s + 0.807s
8. Nicky Hayden Ducati 1m48.798s + 0.826s
9. Michele Pirro Ducati 1m49.034s + 1.062s
10. Aleix Espargaro Aspar Aprilia 1m49.039s + 1.067s
11. Andrea Iannone Pramac Ducati 1m49.135s + 1.163s
12. Marc Marquez Honda 1m49.218s + 1.246s
13. Bradley Smith Tech 3 Yamaha 1m49.257s + 1.285s
14. Hector Barbera Avintia FTR-Kawasaki 1m49.551s + 1.579s
15. Randy de Puniet Aspar Aprilia 1m49.673s + 1.701s
16. Colin Edwards forward FTR-Kawasaki 1m51.101s + 3.129s
17. Danilo Petrucci Ioda-Suter-BMW 1m51.251s + 3.279s
18. Yonny Hernandez PBM Aprilia 1m51.560s + 3.588s
19. Karel Abraham Cardion Aprilia 1m51.619s + 3.647s
20. Claudio Corti Forward FTR-Kawasaki 1m52.189s + 4.217s
21. Lukas Pesek Ioda-Suter-BMW 1m52.226s + 4.254s
22. Michael Laverty PBM-Aprilia 1m52.290s + 4.318s
23. Hiroshi Aoyama Avintia FTR-Kawasaki 1m52.295s + 4.323s
24. Bryan Staring Gresini FTR-Honda 1m53.165s + 5.193s