The class III DNA dependent RNA polymerases (nucleoside triphosphate:RNA nucleotidyltransferase EC 18.104.22.168 from HeLa cells have been solubilized and characterized as to function and properties. Two chromatographically distinct forms of enzyme III, designated polymerases IIIA and IIIB, can be resolved when cell extracts are chromatographed on DEAE-Sephadex columns. Enzymes IIIA and IIIB exhibit nearly identical catalytic properties such as divalent cation stimulation, broad biphasic ammonium sulfate optima, and characteristic alpha-amanitin sensitivities which clearly distinguish them from the homologous enzymes, forms I and II. Polymerases IIIA and IIIB are both primarily localized in the nucleus (greater than 60%). The most notable characteristic of the class III enzymes is a unique sensitivity to inhibition by alpha-amanitin (50% inhibition at 15 mug/ml). HeLa cell enzyme I is not inhibited by the mushroom toxin even at very high concentrations (greater than 400 mug/ml), while HeLa cell polymerase II is inhibited by very low concentrations of amanitin (50% inhibition at 0.003 mug/ml). The three major classes of enzyme (I, II, III) exhibit characteristic sensitivities to alpha-amanitin whether assayed in nuclei, crude homogenates, or in a chromatographically purified state. Using a nuclear in vitro RNA synthesizing system to investigate the alpha-amanitin sensitivities of the synthesis of tRNA precursor (4.5S pre-tRNA) and 5S ribosomal RNA, it was found that the synthesis of these RNA species was inhibited 50% at 15 mug/ml of alpha-amanitin. The alpha-amanitin inhibition curves for the synthesis of pre-tRNA-5S ribosomal RNA in nuclei and the alpha-amanitin titration curves for the partially purified class III enzymes (IIIA and IIIB) are identical. These data, therefore, show that the in vivo functional role of the class III RNA polymerases (IIIA-IIIB) is the transcription of the genes coding for transfer RNA and 5S ribosomal RNA.