We have examined the tumorigenic potential of mitochondrial DNA-depleted (mtDNA-) cells derived from the tumorigenic chicken cell line DU24. The mtDNA- cells were unable to proliferate in the wing web of day-old chicks. Cytoplasmic hybrids resulting from crosses between the mtDNA- whole cells and cytoplasts from enucleated parental cells (mtDNA+) recover both mtDNA and tumorigenicity. These results are in accordance with those obtained in prior experiments where mtDNA was shown to modulate the anchorage-independent phenotype of transformed avian cells.