The hydrogen-evolving reaction of the purified soluble NAD-linked hydrogenase of Alcaligenes eutrophus was used to determine kinetic parameters of the enzyme. The H-2-evolving activity with methyl viologen as electron mediator was 20-fold as compared to that with NADH. In the assay with dithionite-reduced methyl viologen (K-m 0.7 mM) the hydrogenase was most active at a redox potential of - 560 mV and exhibited a pH optimum of 7.0. The K-m for protons, the second substrate for Hz evolution, was 6.2 nM. With electrochemically reduced methyl viologen the pH optimum was shifted to pH 6.0. Double-reciprocal plots of reaction rates versus proton concentrations intercepted at the ordinate for different methyl viologen concentrations. At different pH values such an intercept was also observed with the dye as the varied substrate. The kinetic data are diagnostic for an ordered bisubstrate mechanism where both substrates are bound before the product H-2 is released. Hydrogenase coupled to thylakoid membranes resulted in a constant H-2 evolution rate over 6 h. The system appeared to be limited by the capacity of the thylakoid membranes.