The mitochondrial NADH:ubiquinone oxidoreductase complex (Complex I) is inhibited by N,N'-dicyclohexylcarbodiimide (DCCD), and this inhibition correlates with incorporation of radioactivity from [14C]DCCD into a Complex I subunit of Mr 29,000 (Yagi, T. (1987) Biochemistry 26, 2822-2828). Resolution of [14C]DCCD-labeled Complex I in the presence of NaClO4 showed that the labeled Mr 29,000 subunit was in the hydrophobic fraction of the enzyme. This fraction, which contains greater than 17 unlike polypeptides, was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the Mr 29,000 subunit, containing bound [14C]DCCD, was isolated and purified. The amino acid composition and partial sequence of this subunit corresponded to those predicted from the mitochondrial DNA for the product of the mtDNA gene designated ND-1. The identity of the Mr 29,000 subunit with the ND-1 gene product was further confirmed by immunoblotting and immunoprecipitation experiments, using the hydrophobic fraction of [14C]DCCD-labeled Complex I and antiserum to a C-terminal undecapeptide synthesized on the basis of the human mitochondrial ND-1 nucleotide sequence. Thus, it appears that the DCCD-binding subunits of the respiratory chain Complexes I, III, and IV and in certain organisms the DCCD-binding subunit of the ATP synthase complex (Complex V) are all mtDNA products.