We have studied cultured skin fibroblasts from a patient with a fatal mitochondrial disease manifesting soon after birth. These fibroblasts were found to grow only in the presence of pyruvate and uridine, a characteristic of cells lacking mtDNA (rho0 cells). Southern blot and PCR analyses confirmed that the patient's fibroblasts contained less than 2% of control levels of mtDNA. Biochemical analyses indicated that the activities of all the respiratory-chain enzymes were severely decreased in mitochondria isolated from these fibroblasts. In order to elucidate the underlying molecular defect, cell fusions were performed between enucleated fibroblasts from this patient and a human-derived rho0 cell line (rho0 A549.B2). The resulting cybrids were plated in medium lacking pyruvate and uridine, to select for the restoration of respiratory-chain function. Complementation was observed between the nuclear genome of the rho0 A549.B2 cells and the mtDNA of the patient's cells, restoring mtDNA levels and respiratory-chain function in the cybrid cells. These results indicate that mtDNA depletion in our patient is under the control of the nuclear genome.