Using a whole cell extract from Saccharomyces cerevisiae (bakers' yeast) we have been able to detect a selective RNA polymerase activity originally identified in purified yeast mitochondria (Levens, D., Morimoto, R., and Rabinowitz, M. (1981) J. Biol. Chem. 256, 1466-1473). We have shown that in in vitro transcription reactions this activity recognizes a consensus mitochondrial promoter sequence ATA-TAAGTA (Osinga, K. A., DeHaan, M., Christianson, T., and Tabak, H. F. (1982) Nucleic Acids Res. 10, 7993-8006) in the upstream region of the nuclear GAL10 gene as well as promoters from yeast mitochondrial DNA. Using these promoter-containing templates for in vitro assays, we have chromatographically separated the mitochondrial specific RNA polymerase activity from the three nuclear RNA polymerases (I, II, and III). Further characterization has revealed that this preparation has distinctive properties on two different types of DNA templates, poly[d(AT)] and cloned DNA containing mitochondrial promoters. Salt and divalent cation optima and substrate saturation kinetics are different for the two types of templates. Using promoter-containing DNA as an assay template, we have chromatographically dissociated the RNA polymerase activity into two nonfunctional components. Selective transcription of the GAL10 template is restored when the two components are recombined. It is possible that the RNA polymerase active on poly[d(AT)] is a nonspecific component of the selective transcription apparatus or that two distinct RNA polymerases are present in the preparation.