In vivo, ribosomal RNA of Saccharomyces cerevisiae is transcribed from the light strand of gamma DNA. In vitro, gamma DNA is transcribed with equal efficiency by both of the yeast nuclear RNA polymerases, polymerase I and polymerase II; however the RNA products synthesized by the two enzymes differ from each other both in size and in the relative composition of guanine and cytosine. RNA synthesized by polymerase I hybridizes preferentially to the light strand, while that synthesized by polymerase II hybridizes equally well to either strand. Selective transcription of the light strand of the ribosomal DNA also occurs when high-molecular-weight total nuclear DNA is used as template.