An RNA polymerase was purified 6500-fold to near homogeneity from a whole cell extract of Saccharomyces cerevisiae. The purified enzyme consists of a single 145,000-dalton polypeptide. By subcellular fractionation, the enzyme was localized to the mitochondria. The RNA polymerase activity is alpha-amanitin- and rifampicin-resistant. With single-stranded DNA templates, the enzyme catalyzes the synthesis of polyribonucleotide chains with lengths ranging from less than 10 to greater than 100 residues. It is inactive with double-stranded DNA. Specific antisera inhibit the RNA polymerase activity and recognize the 145,000-dalton polypeptide. The antisera relate this enzyme to previously described yeast mitochondrial RNA polymerase preparations capable of initiation of transcription at mitochondrial promoter sequences (Winkley, C. S., Keller, M. J., and Jaehning, J. A. (1985) J. Biol. Chem. 260, 14214-14223). It therefore appears that the enzyme is a subunit of the yeast mitochondrial RNA polymerase.