Levofloxacin, the L-isomer of ofloxacin, is approximately twice as active as ofloxacin against most Gram-positive and Gram-negative bacteria, and has improved intracellular pharmacokinetic and pharmacodynamic properties. The present work deals with the in-vitro activity of levofloxacin against the obligate intracellular bacteria Rickettsia rickettsii, Rickettsia conorii, 'Israeli spotted fever group rickettsia' (Israeli SFGR) and Coxiella burnetii. Fluoroquinolones, including ofloxacin, have previously been shown to be bacteriostatic against Rickettsia spp. and C. burnetii in vitro. They are reliable alternatives to tetracycline therapy for Mediterranean spotted fever, scrub typhus and acute Q fever. Levofloxacin was bacteriostatic against R. rickettsii, R. conorii and the Israeli SFGR at concentrations of 0.5-1 mg/L, as determined by both a plaque assay and a dye uptake assay. It was also bacteriostatic against C. burnetii isolates, including the Nine Mile, Priscilla and Q212 strains, at concentrations of 0.5-2 mg/L, as determined using the shell vial assay. Overall, levofloxacin could inhibit rickettsial growth at concentrations equal to or half of those necessary for growth inhibition by ofloxacin. Levofloxacin was not bactericidal against C. burnetii at concentrations up to 4 mg/L.