An established avian fibroblast cell line (LSCC-H32) has been found to be inherently resistant to the growth-inhibitory effect of ethidium bromide, when supplied with exogenous uridine. After long-term exposure to ethidium bromide (90 days), the cell population has been transferred to drug-free medium for 60 days, and then seeded at low cell density. Three clones have been isolated and propagated in drug-free medium for 5, 6, and more than 12 months, respectively. It was found that none of these cell lines had detectable cytochrome c oxidase activity and that they were virtually devoid of cytochromes aa3 and b. Mitochondrial DNA was quantitated by DNA-DNA reassociation kinetics with a probe of chicken liver mitochondrial DNA. A mean number of 300 copies of mitochondrial DNA per cell was found in LSCC-H32 cells. Analysis of DNA extracted from cell populations exposed to ethidium bromide for 90 days and then transferred to drug-free medium for long periods of time revealed no mitochondrial DNA molecules by reassociation kinetics or by Southern blot hybridization of HindIII-or AvaI-digested total cellular DNA.