A new DNA polymerase activity, distinct from DNA polymerase gamma, has been identified in bovine heart mitochondria. First detected among proteins isolated in a complex with mitochondrial DNA, the DNA polymerase activity has been partially purified 47,000-fold. Enzyme activity separates from DNA polymerase gamma on several chromatographic columns and appears to copurify with a 38 +/- 2-kDa polypeptide. Unlike DNA polymerase gamma, this enzyme is relatively resistant to inhibition by N-ethylmaleimide and dideoxynucleotides, has moderately low monovalent and high divalent cation requirements, and possesses 20-fold-higher apparent K(m) values for deoxynucleotides. The enzyme polymerizes deoxynucleotides onto a primed template DNA in a relatively nonprocessive fashion and lacks a detectable 3' to 5' exonuclease activity. Many of these characteristics resemble a beta-like mitochondrial DNA polymerase previously identified in, and considered unique to, trypanosomes. We propose that the bovine and trypanosomal enzymes are related and represent a new class of ubiquitous mitochondrial DNA polymerases.