We report the cDNA cloning, chromosomal localization, and a mutation in the human nuclear gene encoding the 18-kD (AQDQ) subunit of the mitochondrial respiratory chain complex I. The cDNA has an open reading frame of 175 amino acids and codes for a protein with a molecular mass of 23.2 kD. Its gene was mapped to chromosome 5. A homozygous 5-bp duplication, destroying a consensus phosphorylation site, in the 18-kD cDNA was found in a complex I–deficient patient. The patient showed normal muscle morphology and a remarkably nonspecific fatal progressive phenotype without increased lactate concentrations in body fluids. The child's parents were heterozygous for the mutation. In 19 other complex I–deficient patients, no mutations were found in the 18-kD gene.