The transmembrane electrical potential (At) generated by Rickettsia prowazekii metabolizing glutamic acid or ATP was determined by flow dialysis with the lipophilic cation tetraphenylphosphonium and with lysine. At pH 7.0, the rickettsiae generated a At as measured by tetraphenylphosphonium distribution of 90 mV. Under similar conditions, cells of R. prowazekii concentrated lysine to a gradient indicating a At of 90 mV. Energy-starved cells of R. prowazekii were able to utilize exogenously supplied ATP as well as glutamic acid to generate a At of 110 mV at pH 8.0. Lysine transport was markedly affected by environmental pH, the optimum pH ranging from 8.0 to 8.5. At as measured with tetraphenylphosphonium was similarly affected in this system, with values ranging from 70 mV at pH 6.0 to 100 mV at pH 8.0. Respiration rates were also affected by the external pH, with a maximum rate of 28 nmol of 02 consumed per min per mg of rickettsial protein occurring at pH 8.0. The pH effects were readily reversible and with a rapid onset.The rickettsiae are gram-negative bacteria that require the cytoplasm of a eucaryotic host cell as an environment for multiplying. Although many investigations have attempted to identify a specific metabolic lesion in these bacteria that accounts for their dependence on the host cell, no such lesion has been detected. However, these studies have yielded much information concerning the metabolic capabilities of the rickettsiae (21,22). Pertinent to the subject of this paper are the observations that rickettsiae synthesize ATP upon the oxidation of glutamate via the tricarboxylic acid cycle by a mechanism sensitive to uncouplers (oxidative phosphorylation) (2,26,27, 33) and actively transport at least one amino acid, lysine (18). These processes have been shown (5-7, 9, 13, 24, 28) in other systems to be dependent on both an intact cytoplasmic membrane and a transmembrane proton electrochemical gradient, the protonmotive force (PMF). The fact that both ATP synthesis and lysine transport are inhibited by protonophores and by high concentrations of the lipophilic cation triphenylmethylphosphonium strongly supports the hypothesis that rickettsiae can generate and maintain a PMF (2,18,27, 33). Although the integrity of the rickettsial cytoplasmic membrane has been questioned, it appears that this component of the rickettsial cell does provide a barrier to certain compounds, is not t Present address: