Honda rider Rea timed his run to perfection, posting the fastest time with just over three minutes of the run-out remaining.
The Ulsterman clocked 1min 31.959secs to top the timesheets after Carlos Checa had paced the previous practice session.
“Going fastest was a pleasant surprise and also kind of our plan," Rea said. "We went out and got on a decent pace right away, sort of 1:32 lap times, but with old tyres. Then we developed a problem with the bike and we lost a bit of time during the session.
"When we did go out again I went out with what I think the race tyre is going to be fitted to the rear, but it did not have the pace of the tyre I used at the end. I did about ten laps on that tyre, all very consistent but it was not going to be the fastest lap of the day.
"I came in to the pits near the end and got a new tyre then went out and we got a bit of a feel-good factor; just to see that we were not that far away from the pace really.”
Checa had again led for much of the way but his progress was halted by a tumble with 20 minutes remaining.
Ahead of Checa were fellow Ducati riders Jakub Smrz and Davide Giugliano with Max Biaggi rounding off the top five.
Kawasaki pair Tom Sykes and Joan Lascorz followed Niccolo Canepa, who impressed for new boys Red Devils Roma Ducati.
There was a scare for Eugene Laverty, who is recovering from a fractured hand but suffered an early fall. He was taken to the medical centre but initial reports suggest he will be fit to race on Saturday.
Leon Haslam, who skipped the opening session, made it out onto the track and completed three laps as he continues to gear up for qualifying on Friday.
Qualifying 1 results:
1. Jonathan Rea GBR Honda WSBK CBR1000RR 1m 31.959s
2. Jakub Smrz CZE Liberty Effenbert Ducati 1098R 1m 32.116s
3. Davide Giugliano ITA Althea Ducati 1098R 1m 32.198s
4. Carlos Checa ESP Althea Ducati 1098R 1m 32.228s
5. Max Biaggi ITA Aprilia Racing RSV-4 1m 32.318s
6. Niccolo Canepa ITA Red Devils Ducati 1098R 1m 32.343s
7. Tom Sykes GBR Kawasaki Racing ZX-10R 1m 32.402s
8. Joan Lascorz ESP Kawasaki Racing ZX-10R 1m 32.443s
9. Marco Melandri ITA BMW Motorrad S1000RR 1m 32.553s
10. Michel Fabrizio ITA BMW Italia Goldbet S1000RR 1m 32.609s
11. Sylvain Guintoli FRA Effenbert Liberty Ducati 1098R 1m 32.644s
12. Leon Camier GBR Crescent Fixi Suzuki GSX-R1000 1m 32.817s
13. Chaz Davies GBR ParkinGO MTC Aprilia RSV-4 1m 32.874s
14. Bryan Staring AUS Pedercini Kawasaki ZX-10R 1m 32.884s
15. Hiroshi Aoyama JPN Honda WSBK CBR1000RR 1m 33.040s
16. Maxime Berger FRA Effenbert Liberty Ducati 1098R 1m 33.075s
17. David Salom ESP Pedercini Kawasaki ZX-10R 1m 33.128s
18. Ayrton Badovini ITA BMW Italia Goldbet S1000RR 1m 33.136s
19. Lorenzo Zanetti ITA PATA Ducati 1098R 1m 33.148s
20. Josh Brookes AUS Crescent Fixi Suzuki GSX-R1000 1m 33.477s
21. Leon Haslam GBR BMW Motorrad S1000RR 1m 33.532s
22. Eugene Laverty IRL Aprilia Racing RSV-4 1m 34.189s
23. Mark Aitchison AUS Grillini Progea BMW S1000RR 1m 34.239s
24. David Johnson AUS Rossair AEP BMW S1000RR 1m 34.855s
25. Raffaele De Rosa ITA Pro Ride Honda CBR1000RR 1m 34.979s