DNA-dependent RNA polymerases were extracted from the nuclei of the poorly differentiated tumor, Morris hepatoma 3924A, and purified by an initial chromatography on a DEAE-Sephadex column followed by fractionation on phosphocellulose and finally on a second DEAE-Sephadex column. Three major forms of RNA polymerase (IA, IB and II) were resolved chromatographically. Enzymes IA, IB and II eluted from DEAE-Sephadex at 75, 150 and 210 mM (NH 4) 2SO 4, respectively. The specific activities (nmol UMP incorporated/mg protein per 15 min) of polymerases IA, IB and II were 40, 43 and 182, respectively. Concurrently, DNA-dependent RNA polymerases were extracted from normal liver and subjected to similar chromatographic procedures. Upon the final DEAE-Sephadex chromatography, enzymes IA, IB and II eluted at 110, 180 and 210 mM (NH 4) 2SO 4, respectively. The recovery of polymerases IA, IB and II after purification was 0.21, 0.28 and 0.42 unit/mg DNA, respectively, for hepatoma enzymes and 0.07, 0.05 and 0.42 unit/mg DNA for the corresponding liver enzymes. Polymerases IA and IB were insensitive to α-amanitin, whereas polymerase II was at least 95% inhibited at low concentrations of the toxin for enzymes from both liver and tumor. The α-amanitin-resistant enzymes from the tumor had very low (NH 4) 2SO 4 optima (12 mM) as compared to the (NH 4) 2SO 4 optima for liver IA and IB (40 mM). Similarly, polymerase II from the hepatoma had a lower (NH 4) 2SO 4 optima (50 mM) than the corresponding liver enzyme (100 mM). Both liver and tumor polymerases IA and IB utilized native and denatured DNA equally well as template whereas polymerase II from either tissue preferentially utilized denatured DNA as template. All enzymes preferred poly[d(A-T)] over calf thymus DNA; a 2–4-fold stimulation was observed with the synthetic polynucleotide. Spermine stimulated transcription of native DNA by polymerases IA, IB and II from liver and hepatoma. Tumor enzymes IA and IB were stimulated 3- and 4.8 fold, respectively, at the optimal concentration of spermine. Tumor polymerase II was stimulated 6 fold by spermine at the optimal concentration of 5.0 mM, at which concentration polymerases IA and IB were inhibited. A significant stimulation with polyamines was observed only when native DNA was used as the template. Spermine inhibited the transcription of poly[d(A-T)] by the three hepatoma enzymes.