The nuclear gene MIP1 is strictly required for mitochondrial DNA replication and mitochondrial DNA polymerase activity (Genga, A., Bianchi, L., and Foury, F. (1986) J. Biol. Chem. 261, 9328-9332). The MIP1 gene was cloned by genetic complementation of the mip1-1 allele after cell transformation with a yeast genomic library and was mapped to the right arm of chromosome XV about 20 centimorgans distal to the cpa1 gene by Southern blot hybridization and tetrad analysis. The mapping of the 5' ends of the MIP1 transcript and the nucleotide sequence analysis of a 4.7-kilobase DNA fragment complementing the mip1-1 allele allowed the determination of an open reading frame of 3762 nucleotides encoding a basic protein of 143.5 kDa. The following data show that the MIP1 gene encodes the catalytic subunit of the replicative mitochondrial DNA polymerase. 1) The mutant ts71 exhibits both a thermosensitive mitochondrial DNA replication in vivo and a thermosensitive mitochondrial DNA polymerase activity is observed, when compared to that of the wild type strain. 3) Chromosomal disruption of the MIP1 gene by an 80% deletion of the gene and its replacement by URA3 gene is not lethal to the cell but elicits total loss of mitochondrial DNA and mitochondrial DNA polymerase activity. 4) The MIP1 protein exhibits sequence similarities with both eukaryotic nuclear DNA polymerases and reverse transcriptases. There is no significant resemblance with prokaryotic DNA polymerases.