Specific transcription of yeast 35 S rDNA by RNA polymerase I has been demonstrated using fractionated extracts prepared from whole cells of Saccharomyces cerevisiae. Determination of the 5'-nucleotides of the in vitro transcripts indicated that two apparent start sites, corresponding to the first (initiating) and fifth nucleotide of the in vivo transcript, were utilized. Production of the 35 S rDNA transcript in this system was not inhibited by alpha-amanitin. Specific transcription of both the 35 S and 5 S rDNA sequences contained on the same template occurred simultaneously in these extracts. Sequential template competition experiments demonstrated that 35 S and 5 S rDNA transcription required different transcription factors. Specific antisera raised against the largest subunit of RNA polymerase I significantly inhibited synthesis of the 35 S rDNA transcript, but had a negligible effect on 5 S rRNA synthesis by RNA polymerase III. Additionally, this 35 S rDNA transcriptional activity was present in extracts prepared from a strain deficient in the mitochondrial RNA polymerase. Experiments using truncated rDNA templates showed that in vitro no more than 206 base pairs of the sequence upstream of the initiation site are required for maximal activity in this system; the enhancer element did not stimulate 35 S rDNA transcription.