Three peaks of DNA-dependent RNA polymerase [EC 2.7.7.6] activity were resolved when the enzyme was prepared from the isolated macronuclei of Tetrahymena pyriformis GL (amicronucleate strain) and chromatographed on DEAE-Sephadex A-25. They were eluted at around 0.05, 0.15, and 0.2 M of ammonium sulfate, and termed TIa, TIb, and TII, respectively. All three enzymes transcribe heat-denatured DNA more efficiently, especially the peak TII, detectable only when heat-denatured DNA was used as a template. Further characterization of each enzyme, after they were rechromatographed on DEAE-Sephadex, demonstrated the similarity in many respects of TIa and TIb, and the distinct nature of the TIa enzyme. TIb, and TII were all insensitive to rifampicin, while only TII was substantially inhibited by α-amanitin. On the other hand, the activity of TII was progressively lowered by increasing the concentration of ammonium sulfate in the assay mixture, a finding incompatible with those obtained thus far. It is concluded from the data that the Tetrahymena polymerase is of eukaryotic and not of bacterial type in spite of the findings indicating the bacterial nature of this organism.