Yeast RNA polymerase B was purified by glycerol density‐gradient centrifugation and phosphocellulose chromatography. The purified enzyme migrates as a main active band on polyacryl‐amide‐gel electrophoresis. No evidence was found in favor of the existence in yeast of two different RNA polymerases B as was the case for calf thymus or rat liver. RNA polymerase activity depends critically on the ionic conditions and on the molecular structure of the template. T4 or T7 phage DNA do not serve as template unless they have been previously denatured. The template activity of calf thymus DNA is drastically reduced after purification on nitrocellulose which eliminates DNA molecules containing single‐stranded regions. RNA chain initiation and elongation was studied with native and denatured calf‐thymus DNA as template. Using denatured DNA, RNA chains are initiated predominantly with GTP and, to a lesser extent, with ATP, whereas almost no chains were initiated with CTP or UTP. The low degree of RNA synthesis obtained with native DNA was due to a deficiency in chain initiation. The preferential transcription of denatured DNA as compared to native DNA is enhanced at low concentrations of nucleoside triphosphates, DNA and enzyme. The effect of various bacterial or mammalian RNA polymerase inhibitors on yeast polymerase was investigated.