A plaque assay system for pathogenic rickettsiae, which utilizes primary chick embryo tissue cultures, is described. It proved to be a highly reproducible measure of infectiousness for Rickettsia rickettsi and R. typhi, which were employed in most studies; as well as for R. canada, R. prowazeki, R. sibirica, R. akari, R. conori, and Coxiella burneti. Plaque-forming units (PFU) were compared to direct rickettsial counts and to 50% infectious dose (ID(50)) values for embryonated eggs, mice, and guinea pigs. Plaque size, appearance, and number were influenced by diluent, incubation temperature after nutrient overlay, centrifugation of inoculated tissue cultures, and number of host cells planted initially in each flask. The most critical factors in plaque formation were diluent used in making rickettsial suspensions and incubation temperature (32 C) after nutrient overlay. Brain Heart Infusion was the only diluent capable of preventing significant delay in plaque formation and decreases in PFU and mouse ID(50). Plaque formation was unaffected by genetic background of host cells, volume of inoculum, temperature and length of incubation period before nutrient overlay, and rapid freezing and thawing of rickettsial seed. Centrifugation of inoculated cultures at 600 x g resulted in 100% irreversible absorption of rickettsiae to host cells within 5 min, whereas without centrifugation at least 4 hr was required to achieve the same effect.