The Fiat Yamaha rider has won the last two races and will start as favourite to make it a hat-trick in his homeland after setting a best lap of one minute 43.259 seconds.
Marlboro Ducati rider Casey Stoner was second while Lorenzo‘s Spanish compatriot and nearest title challenger Dani Pedrosa was third on his Repsol Honda.
Lorenzo leads Pedrosa by 47 points after the opening six rounds and another win in Barcelona this weekend would see him move a step closer to his maiden MotoGP title.
Lorenzo said: “This was a good practice and I was fast straight away again, although I did think that I would be able to improve my lap time a bit more during the session.
“Unfortunately the rear was sliding a lot which was partly to do with the heat, but we will try to improve this tomorrow.
“I love racing here, it’s so close to my home and it’s always extra special for me because all my family and friends come to watch. I hope I can give them a good show.”
Lorenzo is partnered by a new team-mate this weekend as test rider Wataru Yoshikawa begins his three-race contract as replacement for the injured Valentino Rossi. Reigning champion Rossi hopes to be back at Brno in August and the 41-year-old Yoshikawa was way off the pace in 17th and last position.
Rookie Ben Spies continued to impress by guiding his Tech 3 Yamaha to fifth place and the American was pleased with his bike’s performance.
He said: “I’m really surprised I ended so high up in fifth. I went out on one bike and just worked on learning the track and when I switched to the other bike I found the turning was a lot better. I fixed the twitchy feel I was having on the front and that was a positive.
“I didn’t have to hold the front brake to help the bike turn. Now I can let off the brake, throw it into the corner and get it turned.
“I’m looking decent on speed right now but we’ve got to manage it and make sure I’m fast for the end of the race.”