Rhodobacter sphaeroides MDC6522 is able to produce hydrogen (H-2) during photofermentation. Metronidazole (2-methyl-5-nitroimidazole-1-ethanol) has been shown to affect bacterial growth under anaerobic nitrogen-limited conditions in a concentration-dependent manner (within the range of 0.1-2 mM) by increasing lag phase duration and decreasing growth rate. The addition of metronidazole into the growth medium resulted in a delayed decrease of redox potential (E-h): by the addition of 0.1 mM metronidazole E-h decreased to -590 +/- 25 mV, whereas in the presence of 2 mM Eh drop down was to -175 +/- 15 mV. H-2 production during R. sphaeroides growth disappeared in the presence of metronidazole. By addition of 0.5 mM metronidazole H-2 yield was similar to 8 fold lower in comparison with control; whereas the bacterium was unable to produce H-2 in the presence of 1-2 mM. The effects of metronidazole on nitrogenase-dependent H-2 production by R. sphaeroides might be explained by change of general photosynthetic electron transport with metronidazole as an alternative electron acceptor instead of nitrogenase. ATPase activity of membrane vesicles was determined with and without N,N'-dicyclohexylcarbodiimide (DCCD), an inhibitor of the F0F1-ATPase. It was revealed that DCCD-inhibited ATPase activity increased in the presence of metronidazole. It is possible that this effect may be resulted in either by direct affect of metronidazole on the F0F1-ATPase, or by its effect on Eh regulating ATPase activity. Copyright (C) 2013, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.