The Nine Mile, S Q217, and Priscilla isolates, representative of the three major genetic groups of Coxiella burnetii, are known to differ in their susceptibilities to antibiotics. Mechanisms potentially responsible for these differences were investigated. Accumulation of antibiotics by infected L929 cells and purified isolates was measured. In addition, C. burnetii plasmid-transformed Escherichia coli HB101 cells were used to study the possibility that different C. burnetii plasmids are responsible for disparate antibiotic susceptibilities of the isolates. L929 cells recently or persistently infected with the Priscilla isolate exhibited a significantly reduced accumulation of [3H]tetracycline as compared with that in L929 cells infected with either the Nine Mile or S Q217 isolates; accumulation of this drug was greater in cells recently infected each isolate. In contrast, L929 cells recently or persistently infected with the different isolates accumulated [3H]norfloxacin to an equivalent extent. [3H]tetracycline accumulation was approximately the same among the purified isolates. However, as measured by both scintillation and spectrofluorometry, norfloxacin accumulation was significantly diminished in the purified Priscilla isolate. pH had no apparent effect upon isolate permeabilities. The presence of C. burnetii QpH1 or QpRS plasmids did not alter the antibiotic susceptibility of E. coli. Collectively, these results indicate that differential susceptibilities to tetracyclines or fluoroquinolones in C. burnetii isolates may be the result of distinct mechanisms involving altered host-cell (tetracyclines) or isolate-specific (fluoroquinolones) permeabilities.