The photosynthetic bacterium, Rhodobacter capsulatus, produces hydrogen under nitrogen limited, anaerobic, photosynthetic conditions. In the present study, we examine if this photosynthetic bacterium can produce hydrogen under microaerobic conditions in the dark with limiting amounts of O2 and fixed nitrogen. The relationship between hydrogen production, different O2 concentrations and carbon sources as well as two different N-sources, glutamate and ammonium, were studied in batch culture using a Hup- strain of R. capsulatus. Hydrogen production under microaerobic dark fermentation conditions would require that this organism use oxidative phosphorylation instead of photophosphorylation to produce the ATP required for the hydrogen producing enzyme, nitrogenase. The effect of different O2 concentrations, ranging from 0.5% up to 20%, on hydrogen production was examined in dark batch cultures of R. capsulatus grown on RCV medium. Different carbon sources; glucose, lactate, malate, acetate, and succinate were used at various concentrations (20 mM to 40 mM). Likewise, different concentrations of glutamate and ammonium (2 mM to 9 mM) were examined for optimum microaerobic dark hydrogen production. Maximum hydrogen production was observed at an O2 concentration of 4-8%. There was a highly positive correlation between O2 and growth (r²= 0.67). On the other hand, there was a highly negative correlation between O2 and hydrogen productivity (r²= - 0.3). Growth significantly increased initially with increasing O2 but decreased at higher O2 concentrations (p< 0.01). Succinate (25mM) together with glutamate (3.5mM) gave the highest hydrogen production [6.0 umole hydrogen/(mg CDW/ml)] whereas lactate gave a broad maximum hydrogen production over different lactate concentrations. The implications for developing a system capable of high H2 productivity are discussed.