Mechanisms of resistance to quinolones were investigated in two in vitro DNA gyrase mutant Coxiella burnetii strains. Accumulation of pefloxacin was found to be lower in resistant strains than in susceptible strains. Pre-incubation with carbonyl cyanide m-chlorophenylhydrazone had no apparent effect on intracellular quinolone fluorescence, indicating that an active efflux mechanism is not responsible for the decrease of pefloxacin accumulation. No differences were detected between the strains after outer membrane protein analysis. Decreased accumulation was found in all strains at acidic pH. It is possible that, apart from the mutation-induced mechanism of resistance, a pH-related mechanism is involved in the drug accumulation process.