An NAD-dependent 5,10-methylenetetrahydrofolate dehydrogenase has been purified to homogeneity from autotrophically and heterotrophically grown cells of Acetobacterium woodii. The enzymes from the differently grown cells were indistinguishable by gel filtration and sodium dodecyl sulfate electrophoresis and have a final specific activity of 670 units mg-1. The enzyme is oxygen-labile; therefore, it was isolated under anaerobic conditions in the presence of dithiothreitol. The oxidized enzyme can be reactivated with 5 mM dithiothreitol, the half-time of activation being 19 min. The forward and reverse reaction initial velocity kinetics was studied and the enzyme was found to follow a substituted (ping-pong) reaction mechanism. With this model, the Km values for NAD and 5,10-methylenetetrahydrofolate are 4.0 and 0.26 mM, while for NADH and 5,10-methenyltetrahydrofolate, they are 2.0 and 1.0 mM, respectively. The equilibrium constant at pH 6.7, determined by the Haldane relationship, is approximately equal to 2.0, favoring the formation of NADH and 5,10-methenyltetrahydrofolate. The purified enzyme is a Mr = 55,000 dimer which lacks 10-formyltetrahydrofolate synthetase and 5,10-methenyltetrahydrofolate cyclohydrolase activities. At pH 6.7, the conversion of 5,10-methylenetetrahydrofolate to 5,10-methenyltetrahydrofolate occurs at a rate of 98,600 mol min-1 mol-1 of enzyme, while the reverse reaction occurs at a rate of 95,600 mol min-1 mol-1 of enzyme.