The effects of γ-irradiation of chromatin on its function as a template for the synthesis of RNA by DNA-dependent RNA polymerase have been studied. Chromatin extracted from calf thymus and from rat liver, and RNA polymerase extracted from E. coli were used. The initial effect of irradiation is an enhancement of the template activity of chromatin. Kinetic studies suggest that the enhancement is not attributable to an increase in the number of enzyme binding sites on the chromatin available for transcription. At high doses, priming ability is inhibited. This inhibition is accompanied by an increased number of binding sites, but the polymerase bound to these newly created sites is apparently inactive in RNA synthesis. A complete nearest neighbor base frequency analysis of RNA synthesized on chromatin templates has been made. The base composition and nearest neighbor frequencies of the RNA product indicate that the transcription from chromatin is asymmetric. With irradiated chromatin template, changes in base composition of the synthesized RNA were found which may be attributable to a greater radiation sensitivity of the adenine-thymine base pair in the chromatin DNA.