At least two enzymes that phosphorylate diphosphopyridine nucleotides were detected in Saccharomyces cerevisiae: NADH-specific kinase was localized exclusively in the mitochondria, and NAD⁺-specific kinase was distributed in the microsomal and cytosol fractions but not in the mitochondria. The identity of NAD⁺ kinase detected in the two fractions remains equivocal. NADH kinase was highly purified 1, 041-fold from the mitochondrial fraction. The K_m values for NADH and ATP were 105μM and 2.1mM, respectively. The relative molecular mass was estimated to be 160, 000 by means of molecular sieve chromatography. From inactivation studies with SH inhibitors and protection by NADH, it was demonstrated that a cysteine residue is involved in the binding site of NADH.