Mitochondrial DNA polymerase from Saccharomyces cerevisiae, purified 3500 fold, was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis into three polypeptides. The major 150 kDa polypeptide was probably the catalytic subunit of the mitochondrial (mt) DNA polymerase and the other two polypeptides could be either proteolytic cleavage products of the polymerase, other subunits of the enzyme or protein contaminants. The mtDNA polymerase preferred an A+T-rich DNA template and did not require any RNA primer for DNA synthesis, at least under in vitro reaction conditions. It showed higher processivity on a double-stranded linear DNA template than on a single-stranded circular DNA template, and was capable of synthesizing at least about 1200 nucleotide primer-extended products without any major pause on a double-stranded DNA template.