Rhodobacter sphaeroides MDC 6521 isolated from Arzni mineral springs in Armenia is able to produce bio-hydrogen (H-2) in anaerobic conditions upon illumination in the presence of various metal ions. The significant aspect in regulation of H-2 production by these bacteria and its energetics is the requirement for F0F1-ATPase, the main membrane enzyme responsible for generation of proton motive force under anaerobic conditions. In order to determine the mediatory role of F0F1 in H-2 production, the effects of various metal ions, (Mn2+, Mg2+, Fe2+, Ni2+, and Mo6+) on N,N'-dicyclohexylcarbodiimide inhibited ATPase activity of R. sphaeroides membrane vesicles were investigated. These ions in appropriate concentrations considerably enhanced H-2 production, which was not observed in the absence of Fe2+, indicate the requirement for Fe2+. The R. sphaeroides membrane vesicles demonstrated significant ATPase activity. In the absence of Fe2+ inhibition (80 mu M) of ATPase activity was observed, which was increased by addition of metal ions. A higher ATPase activity was detected in the presence of Fe2+ (80 mu M) and Mo6+ (16 mu M). These results indicate a relationship between the F0F1-ATPase activity and H-2 production that might be a significant pathway to provide novel evidence of a requirement for F0F1-ATPase in H-2 production by R. sphaeroides. Copyright (c) 2012, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.