Karlovic broke his previous ATP record, but still lost in a gruelling five-set match to Stepanek.
His next opponent with be American Tim Smyczek, who beat big-serving Croat Ivo Karlovic 6-4 7-6 (7-5) 7-5.
Potential third- and fourth-round opponents Ivo Karlovic and Stanislas Wawrinka suffered surprise defeats.
In total, Stepanek and Karlovic played out 81 games before the first break of serve at 14-14 in the decider.
Meanwhile in Hyogo, Croatia lead Japan 2-1 after Ivo Karlovic and Ivan Dodig beat Tatsuma Ito and Yuichi Sugita 6-4, 6-4, 3-6, 6-3.
Ivo Karlovic, the 6-foot-10 Croatian who was defeated in the second round by Mr. Murray, took five towels during his rain-delayed first-round match.
Murray never looked like following Karlovic and Wawrinka out of the tournament at the first hurdle, despite occasional moments of brilliance from the talented Gulbis.
American James Blake lost 4-6 7-6 6-4 to Croatia's Ivo Karlovic, a defeat that gives Andy Murray renewed hope of reaching the season-ending Masters Cup.
The Argentine pair triumphed 3-6 7-6 6-3 6-7 8-6 against Marin Cilic and Ivo Karlovic in a match lasting one minute shy of five hours.
The 28-year-old Davydenko, who won the Paris title in 2006, will next play either big-serving Croatian Ivo Karlovic or fellow London hopeful, ninth-seeded Swede Robin Soderling.
During that time, Karlovic had sent down 78 aces, 23 more than his previous record in losing in this year's French Open first round to Lleyton Hewitt.
Together with Mario Ancic, Goran Ivanisevic and Ivo Karlovic, Ljubicic was part of Croatia's victorious 2005 Davis Cup team, becoming the only unseeded country to win the event.
Swedish ninth seed Robin Soderling kept alive his hopes of snatching a place at the ATP World Tour Finals in London by defeating Croatia's Ivo Karlovic 6-4 7-6.
Isner, with 98, and Mahut (95) have also both smashed the previous record for the most aces, the 78 set by Ivo Karlovic in a Davis Cup tie in 2009.
World number two Novak Djokovic gave the visitors the advantage with a 7-6 (7-3), 6-4, 6-1 over Ivan Ljubicic who came out of Davis Cup retirement as cover for the injured Ivo Karlovic.
What determines whether you play a dangerous floater like huge serving Ivo Karlovic, who owns wins over Roger Federer and Novak Djokovic, or a qualifier like Bradley Klahn, ranked 489 in the world?
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Federer , seeking his fifth title in the season's opening grand slam and his 17th overall, will face big-serving Croatian Ivo Karlovic in the last 32 following Beck's pre-match withdrawal with a back injury.
Stepanek and Berdych's triumph came on the back of singles victories for the pair on Friday when the Czech Republic opened up a 2-0 first-day lead in the semi-final, despite Ivo Karlovic firing a record 78 aces.
That tie is nicely poised after Croatian No.1 Marin Cilic beat David Nalbandian 5-7 6-4 4-6 7-6 6-3 before Juan Martin del Potro hit back leveling the match with a straight sets victory over Ivo Karlovic 6-2 7-6 6-1.
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