Mitochondrial transcription factor A (mtTFA) is a key activator of mitochondrial transcription in mammals. It also has a role in mitochondrial DNA (mtDNA) replication, since transcription generates an RNA primer necessary for initiation of mtDNA replication. In the mouse, testis-specific mtTFA transcripts encode a protein isoform that is imported to the nucleus rather than into mitochondria of spermatocytes and elongating spermatids. We now report molecular characterization of human mtTFA (h-mtTFA) expression in somatic tissues and male germ cells. Similarly to the mouse, analysis of cDNAs and Northern blots identified abundant testis-specific transcript isoforms generated by use of alternate transcription initiation sites. However, unlike the mouse, none of the testis-specific transcripts predicts a nuclear protein isoform, and Western blot analysis identified only the mitochondrial form of h-mtTFA in human testis. Immunohistochemistry and in situ were used to compare the distribution of mtTFA protein, testis-specific mtTFA transcripts, mtDNA and mtRNA in sections of human testis. Our results show that the mtTFA protein and mtDNA exhibit parallel gradients with high levels in undifferentiated male germ cells and low levels or an absence in different male germ cells. Testis-specific transcripts exhibit the opposite pattern, suggesting that in both humans and mice, these testis-specific mtTFA transcripts down-regulate mtTFA protein levels in mammalian mitochondria. Our findings demonstrate that mtTFA does not have a critical role in the nucleus, suggest a mechanism for reducing mtDNA copy number during spermatogenesis and have implications for the understanding of maternal transmission of mtDNA.