Purple non-sulfur photosynthetic bacteria such as Rhodobacter sphaeroides and Rhodopseudomonas palustris produce hydrogen gas (H-2) via proton reduction, which is catalyzed by nitrogenase. Although the expression of nitrogenase is usually repressed under nitrogen-sufficient conditions, a partial deletion of nifA, which encodes a transcriptional activator of nitrogen-fixation genes, has been reported to enable the constitutive expression of nitrogenase in R. palustris. In this study, we evaluated the effects of a similar mutation (nifA* mutation) on H-2 production during the photoheterotrophic growth of R. sphaeroides, based on the notion that H-2 production by nitrogenase compensates for the loss of CO2 fixation via the Calvin cycle, thereby restoring the redox balance. The chromosomal nifA* mutation resulted in the slight restoration of the photoheterotrophic growth of a mutant strain lacking ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO), the key enzyme of the Calvin cycle, when the strain was cultured in van Niel's yeast medium. In addition, the strain with the nifA* mutation produced detectable levels of H-2 during photoheterotrophic growth with acetate and ammonium; however, the H-2 production was considerably lower than that observed during the photoheterotrophic growth of the strain with acetate and l-glutamate, where l-glutamate serves as a poor nitrogen source, thereby causing nitrogenase derepression. On the other hand, introduction of a multicopy plasmid harboring nifA* markedly restored the photoheterotrophic growth of the RubisCO-deletion mutant in van Niel's yeast medium and resulted in efficient H-2 production during the photoheterotrophic growth with acetate and ammonium.