Coenzyme Q₁₀ is a mobile lipophilic electron carrier located in the inner mitochondrial membrane. Defects of coenzyme Q₁₀ biosynthesis represent one of the few treatable mitochondrial diseases. We genotyped a patient with primary coenzyme Q₁₀ deficiency who presented with neonatal lactic acidosis and later developed multisytem disease including intractable seizures, global developmental delay, hypertrophic cardiomyopathy, and renal tubular dysfunction. Cultured skin fibroblasts from the patient had a coenzyme Q₁₀ biosynthetic rate of 11% of normal controls and accumulated an abnormal metabolite that we believe to be a biosynthetic intermediate. In view of the rarity of coenzyme Q₁₀ deficiency, we hypothesized that the disease-causing gene might lie in a region of ancestral homozygosity by descent. Data from an Illumina HumanHap550 array were analyzed with BeadStudio software. Sixteen regions of homozygosity >1.5 Mb were identified in the affected infant. Two of these regions included the loci of two of 16 candidate genes implicated in human coenzyme Q₁₀ biosynthesis. Sequence analysis demonstrated a homozygous stop mutation affecting a highly conserved residue of COQ9, leading to the truncation of 75 amino acids. Site-directed mutagenesis targeting the equivalent residue in the yeast Saccharomyces cerevisiae abolished respiratory growth.