The nuclear-encoded DNA polymerase gamma (DNA POL gamma) is the sole DNA polymerase required for the replication of the mitochondrial DNA. We have cloned the cDNA for human DNA POL gamma and have mapped the gene to the chromosomal location 15q24. Additionally, the DNA POL gamma gene from Drosophila melanogaster and a partial cDNA for DNA POL gamma from Gallus gallus have been cloned. The predicted human DNA POL gamma polypeptide is 1239 amino acids, with a calculated molecular mass of 139.5 kDa. The human amino acid sequence is 41.6, 43.0, 48.7, and 77.6% identical to those of Schizosaccharomyces pombe, Saccharomyces cerevisiae, Drosophila melanogaster, and the C-terminal half of G. gallus, respectively. Polyclonal antibodies raised against the polymerase portion of the protein reacted specifically with a 140-kDa protein in mitochondrial extracts and immunoprecipitated a protein with DNA POL gamma like activity from mitochondrial extracts. The human DNA POL gamma is unique in that the first exon of the gene contains a CAG10 trinucleotide repeat.