In order to examine the mediatory role of proton motive force (∆p) or proton ATPase in H₂ production by Rhodobacter sphaeroides, ∆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 (∆φ) was measured from the distribution of tetraphenylphosphonium cation; pH gradient (∆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, ∆φ was of -94 mV and the reversed ∆pH was +30 mV, resulting in ∆p of -64 mV. The addition of N,N'-dicyclohexylcarbodiimide (DCCD), the F₀F₁-ATPase inhibitor, was not affect ∆φ. It was shown that ∆φ varies nearly linearly with ΔpH, ∆φ 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 ∆pH, resulting in a low ∆p under anaerobic-dark conditions. Intracellular ATP concentrations and energetic charge (EC) were measured to evaluate a metabolism activity of R. sphaeroides.