A factor which eliminated nonspecific transcription of cloned rat rDNA was extensively purified from rat mammary adenocarcinoma ascites cells by successive fractionations on DEAE-Sephadex and heparin-Sepharose columns. The fractions containing RNA polymerase I (HS-B) and fractions eluting thereafter (HS-C) from the heparin-Sepharose column were pooled separately. Addition of HS-C to HS-B prevented random transcription of rDNA and yielded an accurate rDNA transcript with negligible non-specific transcription. The factor was essentially homogeneous and corresponded to Poly(ADP-ribose) polymerase with respect to molecular weight, dependence on DNA for its activity and its ability to undergo auto ADP-ribosylation. The total amount of protein in the transcription assay was approximately 2 micrograms, which indicates a high degree of purity of all the factors required for specific transcription of rDNA.