Inducer, inhibitor, and mutant studies on three hydrogenase activities of Rhodospirillum rubrum indicate that they are mediated by three distinct hydrogenase enzymes. Uptake hydrogenase mediates H-2 uptake to an unknown physiological acceptor or methylene blue and is maximally synthesized during autotrophic growth in light. Formate-linked hydrogenase is synthesized primarily during growth in darkness or when light becomes limiting, and links formate oxidation to H-2 production. Carbon-monoxide-linked hydrogenase is induced whenever CO is present and couples CO oxidation to H-2 evolution. The enzymes can be expressed singly or conjointly depending on growth conditions, and the inhibitor or inducer added. All three hydrogenases can use methyl viologen as the mediator for both the H-2 evolution and H-2 uptake reactions while displaying distinct pH optima, reversibility, and sensitivity to C2H2, gas. Yet, we present evidence that the CO-linked hydrogenase, unlike the uptake hydrogenase, does not link to methylene blue as the electron acceptor. These differences allow conditions to be established to quantitatively assay each hydrogenase independently of the others both in vivo and in vitro.