Abstract Methylenetetrahydrofolate dehydrogenase (5,10-methylenetetrahydrofolate: NADP oxidoreductase EC 1.5.1.5) has been purified from Clostridium thermoaceticum, an obligate anaerobic thermophile. The enzyme has a molecular weight of 55,000 ± 5,000 and consists of 2 subunits of equal molecular weight. The sedimentation constant s20,w0 is 3.8 S, the partial specific volume 0.752 cc per g, and the Stokes radius 31.7 A. The enzyme appears homogeneous during Sephadex chromatography and ultracentrifugation, but during disc gel electrophoresis at pH 8.9 two bands of equal intensity are seen. Both bands are enzymatically active, and, when isolated individually, they again show the same two bands in the electrophoresis, indicating an equilibrium between the two forms. The enzyme has high thermal stability with a temperature optimum above 64° and exhibits a broken line in an Arrhenius graph. The apparent Km for NADP is 9 x 10-5 m and for methylenetetrahydrofolate 3.5 x 10-5 m. The enzyme is specific for NADP and NAD is a competitive inhibitor with a Ki of 2 x 10-4 m.