The NAD+-NADP+-dependent 2,4-diaminopentanoic acid C4 dehydrogenase from Clostridium sticklandii has been purified to homogeneity by the criteria of disc gel electrophoresis and ultracentrifugation. The weight average molecular weight of the native enzyme as determined by high speed sedimentation equilibrium is 72,000. Sedimentation velocity indicated an s20,w of 4.47 S. Sodium dodecyl sulfate disc gel electrophoresis and high speed sedimentation in 6 m guanidine HCl established that the enzyme is composed of 2 subunits of identical size. The enzyme is sensitive to thiol inhibitors, and titration with 5,5'-dithiobis(2-nitrobenzoic acid) and p-chloromercuribenzoate demonstrated the presence of six sulfhydryl groups per mole. Amino acid analysis indicated that the enzyme contains 6 half-cystine residues per mole.