Penicillin G (greater than or equal to 20 micrograms/ml) is rapidly rickettsiacidal for intracellular Rickettsia prowazekii. Light and electron microscopic examinations revealed that penicillin G in culture medium induced a predictable transformation into typical enlarging spheroplasts deficient in the internal, putative peptidoglycan layer of the outer membrane. Under certain conditions, spheroplasts ruptured to discharge contents into host cell cytoplasm and to leave empty shells of defective outer membrane and diffuse amorphous intracytoplasmic antigen. Host cell destruction often accompanied spheroplast rupture. Penicillin G (100 micrograms/ml) caused similar spheroplast formation by Rickettsia rickettsii, but 1,000 micrograms/ml caused neither growth inhibition nor spheroplast formation in Rickettsia tsutsugamushi. The clinical and epidemiological significance of a practical rickettsiacidal drug for the treatment of louse-borne typhus fever is discussed. Practical pharmacologic considerations preclude the use of penicillin for the treatment of typhus or spotted fever.