That allowed Rossi's team-mate Nicky Hayden to inherit the position, having spent much of the race just ahead of fellow American Colin Edwards.
Edwards' Monster Yamaha Tech 3 team-mate Cal Crutchlow joined Pedrosa and Bautista on the DNF list, as did fellow rookie Karel Abraham.
Abraham, whose father and team manager owns the Brno team, was unable to finish his home race after an off-track moment was followed by an engine failure.
Bautista's wild-card team-mate John Hopkins was forced to withdraw from the event after fracturing fingers in his right hand during Saturday morning practice.
A test session will be held at the circuit on Monday, which will feature next year's 1000cc bikes from Honda and Yamaha.
Czech Republic Grand Prix:
12. de Puniet