Two human cell lines (termed ρ⁰), which had been completely depleted of mitochondrial DNA (mtDNA) by long-term exposure to ethidium bromide, were found to be dependent on uridine and pyruvate for growth because of the absence of a functional respiratory chain. Loss of either of these two metabolic requirements was used as a selectable marker for the repopulation of ρ⁰ cells with exogenous mitochondria by complementation. Transformants obtained with various mitochondrial donors exhibited a respiratory phenotype that was in most cases distinct from that of the ρ⁰ parent or the donor, indicating that the genotypes of the mitochondrial and nuclear genomes as well as their specific interactions play a role in the respiratory competence of a cell.