Rickettsiae grow only intracellularly, and the antibiotic susceptibilities of these bacteria have been assessed by either plaque, dye uptake, or immunofluorescence assays, which are time-consuming. We used a quantitative PCR (with the LightCycler instrument) to assess the levels of inhibition of Rickettisa felis, R. conorii, and R. typhi DNA synthesis in the presence of various antibiotics. We established the kinetics of rickettsial DNA during growth and showed that R. conorii grows more quickly than R. typhi in cell culture, with maximum replication occurring after 5 and 7 days, respectively. The MICs of the antibiotics tested for R. conorii and R. typhi by the quantitative PCR assay were similar to those previously obtained by plaque and dye uptake assays. We found that R. felis is susceptible to doxycycline, rifampin, thiamphenicol, and fluoroquinolones but not to gentamicin, erythromycin, amoxicillin, or trimethoprim-sulfamethoxazole. The resistance of this new species to erythromycin is consistent with its current taxonomic position within the spotted fever group. We believe that quantitative PCR could be used in the future to simplify and shorten antibiotic susceptibility assays of other rickettsiae and other strict intracellular pathogens.