Arylazido-beta-alanyl NAD+ (A3'-O-(3-[N-(4-azido-2-nitrophenyl)amino]propionyl) NAD+) is a potent competitive inhibitor with respect to NADH (apparent Ki, 1.7-2.7 microM) for the purified mitochondrial NADH dehydrogenase (EC 184.108.40.206). Upon photoirradiation of the dehydrogenase in the presence of arylazido-beta-[3-3H]-alanyl NAD+, radioactivity was found to be associated with the Mr = 57,000 subunit without significant labeling of what are considered to be the enzyme's two smaller subunits. This labeling could be prevented by the presence of NADH during photoirradiation. In contrast to arylazido-beta-alanyl NAD+, arylazido-beta-alanyl NADP+ (N3'-O-(3-[N-(4-azido-2-nitrophenyl)amino]-propionyl) NADP+) did not inhibit dehydrogenase activity nor did photoirradiation of the enzyme in the presence of the radiolabeled analogue (arylazido-beta-[3-3H]alanyl NADP+) result in radioactivity being incorporated into the enzyme. It is concluded that the Mr -- 57,000 subunit contains the pyridine nucleotide-binding site of the mitochondrial NADH electron transport system.