Casey Stoner took his second pole in a row for Repsol Honda despite running on and dropping his RCV in the gravel at turn one on his last lap.
Team-mate Dani Pedrosa claimed second from Yamaha's Jorge Lorenzo on his final lap.
Valentino Rossi, Hiroshi Aoyama, Ben Spies, Colin Edwards, Karel Abraham and Randy de Puniet all suffered lowside falls during the hour.
Pedrosa will undergo shoulder surgery early next week to try and cure his left-arm numbness.
Injured Suzuki rider Alvaro Bautista has been replaced by John Hopkins for this event. Hopkins is making his first grand prix appearance since Valencia 2008.
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1. Casey Stoner
AUS Repsol Honda Team 1m 38.757s
2. Dani Pedrosa
ESP Repsol Honda Team 1m 38.915s
3. Jorge Lorenzo
ESP Yamaha Factory Racing 1m 38.918s
4. Ben Spies
USA Yamaha Factory Racing 1m 39.390s
5. Marco Simoncelli
ITA San Carlo Honda Gresini 1m 39.486s
6. Andrea Dovizioso
ITA Repsol Honda Team 1m 39.709s
7. Randy de Puniet
FRA Pramac Racing Team 1m 39.892s
8. Colin Edwards
USA Monster Yamaha Tech 3 1m 39.895s
9. Cal Crutchlow
GBR Monster Yamaha Tech 3 1m 40.019s
10. Hiroshi Aoyama
JPN San Carlo Honda Gresini 1m 40.168s
11. Nicky Hayden
USA Ducati Marlboro Team 1m 40.175s
12. Valentino Rossi
ITA Ducati Marlboro Team 1m 40.185s
13. Hector Barbera
ESP Mapfre Aspar Team 1m 40.217s
14. John Hopkins
USA Rizla Suzuki MotoGP 1m 40.310s
15. Loris Capirossi
ITA Pramac Racing Team 1m 40.523s
16. Karel Abraham
CZE Cardion AB Motoracing 1m 40.601s
17. Toni Elias
ESP LCR Honda MotoGP 1m 41.114s
Fastest practice time:
ESP Repsol Honda 1m 38.864s
2010 Spanish Grand Prix (May 2):
ESP Repsol Honda 1m 39.202s
Fastest race lap:
ESP Repsol Honda 1m 39.731s