Mitochondrial DNA (mtDNA) is highly susceptible to mutations due to the low level of DNA repair and the presence of a high level of reactive oxygen species in the organelle. Although mtDNA mutations have been implicated in degenerating diseases, aging, and cancer, very little is known about the role of T cells in immunosurveillance for mtDNA aberrations. Here, we describe T-cell recognition of a peptide translated from an alternative open reading frame of the mitochondrial cytochrome b (cyt b) gene in melanoma cells established from a patient. To understand how the cyt b gene is transcribed and translated in tumor cells, we found that cyt b-specific CD4(+) T cells only recognized protein fractions derived from cytoplasm and not from mitochondria. However, T-cell recognition of tumor cells could be inhibited by treatment of tumor cells with rhodamine 6G inhibitor, which depletes mitochondria. These findings suggest that cyt b mRNA is leaked out of the mitochondria and then translated in the cytoplasm for presentation to CD4(+) T cells. The cyt b cDNAs from this patient contain highly heteroplasmic transition mutations compared with control cell lines, suggesting a compromise of mitochondrial integrity that may have contributed to melanoma induction or progression. These findings provide the first example of a mitochondrial immune target for CD4(+) T cells and therefore have implications for the immunosurveillance of mitochondrial aberrations in cancer patients.