Suspensions of partially purified Rickettsia prowazekii yielded two bands of organisms when centrifuged to equilibrium in Renografin density gradients. Rickettsiae from the lower, heavy band were defective in their infective and metabolic activities, as compared to organisms from the light band. The greater density in Renografin of heavy-banding organisms was due to their lack of permeability barrier to it, as evidenced by the absence of plasmolysis in hypertonic Renografin. In contrast, light-banding rickettsiae were able to exclude Renografin, since they were plasmolyzed in it. The proportion of heavy-banding organisms in a rickettsial suspension was influenced by the growth phase they were in when harvested from infected yolk sacs, as well as by the conditions and media to which they subsequently were exposed. We have concluded that these defective forms arise from the degeneration of light-banding rickettsiae. This separation of two functional classes of rickettsiae in Renografin density gradients has been exploited (i) to increase the uniformity of the suspensions by removing many noninfectious particles and (ii) to determine rapidly the integrity of certain properties of the cytoplasmic membrane of organisms exposed to a variety of conditions.