Saccharomyces cerevisiae mtDNA polymerase, isolated as a single 135-kDa recombinant polypeptide, showed high processivity and a capacity of use poly(dA).oligo(dT), poly(rA).oligo(dT), or primed bacteriophage M13 DNA as a template. In a primer extension assay, the enzyme exhibited an intrinsic 3'-5'-exonuclease activity. By optimizing the polymerization reaction conditions, apparent Km and Vmax values could be determined for the incorporation of dTTP, 2'-3'-dideoxy-TTP (ddTTP), 3'-azido-TTP (AZTTP), 3'-fluoro-TTP, dCTP, 2'-3'-dideoxy-CTP, and didehydro(d4)CTP. The yeast mtDNA polymerase used ddTTP, 3'-fluoro-TTP, and ddCTP almost as efficiently as natural deoxynucleoside trisphosphates. Both 3'AZTTP and d4CTP were each significantly less efficient as substrates. Overall, the kinetic data with mtDNA polymerase were very similar to those of the recombinant human immunodeficiency virus reverse transcriptase control. Terminally incorporated AZTTP or ddTTP was not removed by the 3'-5' exonuclease activity of mtDNA polymerase. This may explain the inhibition of mtDNA replication observed in anti-human immunodeficiency virus treatment with dideoxynucleoside analogs for their effects of mtDNA polymerase could be of value in future rational drug design.