We have investigated the photoreduction of pyridine nucleotides by crude extracts and chromatophores of Rhodopseudomonas spheroides.Our findings are as follows:NADP is preferentially photoreduced by crude extracts (37,000 x g supernatant fraction) and there is no requirement for the addition of exogenous substrates. Crude extracts also catalyze a nonphotosensitive reduction of NAD.NADP photoreduction is completely inhibited if an NADH trapping system is present and indicates that NADH is required for NADP photoreduction.Washed chromatophores (150,000 x g pellet) do not catalyze NADP photoreduction unless the supernatant fraction is added. The restoring effect of supernatant fraction is lost upon boiling and dialysis. However, supernatant materials can be replaced by an NADH generating system. There is no requirement for anaerobic conditions.Evidence has been presented which suggests that Rhodopseudomonas spheroides contains an energy-linked transhydrogenase that can be driven by a high energy intermediate generated by light or ATP. This intermediate may also be functional in ATP synthesis. The synthesis of ATP and the ATP-supported transhydrogenase is inhibited by oligomycin. This inhibitor does not affect the light-mediated reaction.