In order to examine the mediatory role of proton motive force (Delta p) or proton ATPase in H-2 production by Rhodobacter sphaeroides, Delta p was determined under anaerobic conditions in the dark, and the ATPase activity has been studied in R. sphaeroides strain A-10, isolated from Arzni mineral springs in Armenia. Membrane potential (Delta phi) was measured from the distribution of tetraphenylphosphonium cation; pH gradient (Delta pH) was the difference between the external and cytoplasmic pH values, and the latter was measured by 9-aminoacridine (9-AA) fluorescence changes. At pH 7.5, Delta phi was of -94 mV and the reversed Delta pH was +30 mV, resulting in Delta p of -64 mV. The addition of N,N'-dicyclohexylcarbodiimide (DCCD), the F0F1-ATPase inhibitor, was not affect Delta phi. It was shown that Delta phi varies nearly linearly with Delta pH, Delta phi increased from -57.1 mV at pH 6.0 to -103.8 mV at pH 8.0; it was compensated at high external pH by a reversed Delta pH, resulting in a low Delta p under anaerobic-dark conditions. Intracellular ATP concentrations and energetic charge (EC) were measured to evaluate a metabolism activity of R. sphaeroides.