A rapid procedure for the simultaneous purification of yeast RNA polymerases I, II, and III is described. The procedure involves direct fractionation of a yeast cell extract by heparin agarose affinity chromatography, followed by glycerol gradient centrifugation and DEAE-Sephadex chromatography. The purification can be completed in 3-4 days using 20-200 g of yeast cells. Two forms each of RNA polymerases I, II, and III are resolved after DEAE-Sephadex chromatography. In the cases of RNA polymerases I and II, these forms differ in subunit structure. The transcriptional properties of the isolated enzymes were determined using hybrid plasmid DNA templates containing yeast ribosomal and glycolytic structural genes. Both forms of RNA polymerases I and II transcribe plasmid DNA templates with low efficiency and no evidence for selective initiation of transcription was found for these enzymes using a wide variety of templates. Both forms of RNA polymerase III transcribe plasmid DNA templates with high efficiency and direct the synthesis of discrete transcripts. Sites for initiation and termination of transcription by RNA polymerase III within defined plasmid DNA templates were determined. The data show that RNA polymerase III-dependent synthesis of discrete transcripts from restriction endonuclease-digested plasmid DNA templates is initiated from selected ends of the templates and terminates at discrete sites downstream from the site of initiation. RNA polymerase III initiates synthesis at many sites within supercoiled plasmid DNA templates.