Rickettsia typhi cultivated in the yolk sac of chicken embryos or in L cells irradiated 7 days previously was separated from host cell components by two cycles of Renografin density gradient centrifugation. Preliminary steps involved differential centrifugation and centrifugation over a layer of 10% bovine plasma albumin of infected yolk sac suspensions, or trypsinization and passage through filters of wide porosity of infected L cell suspensions. Rickettsial preparations obtained by these methods appeared to be free from host cell components while retaining high levels of hemolytic activity, egg infectivity, and capacity to catabolize glutamate. Average yields were 3.3 mg of rickettsial protein per yolk sac or 0.44 mg per 16-oz (ca. 475-ml) L cell culture. Extracts from these two preparations displayed malate dehydrogenase activity of electrophoretic mobility identical to each other but quite different in migration patterns from the corresponding host cell enzymes. This method of separation of rickettsiae from host cell constituents appears to be particularly well suited for the study of rickettsial enzymatic activity.