The 5' end of the RNA transcript of the Saccharomyces cerevisiae RNA polymerase related gene, RPO31, was localized on the RPO31 locus using Northern analyses and primer extension experiments. The amino terminal protein sequence was determined for the largest (relative mass approximately 160,000) subunit polypeptide of S. cerevisiae RNA polymerase III. The N-terminal amino acid sequence of this polypeptide was shown to be identical to that predicted for a putative RPO31 polypeptide initiated at the first ATG codon located 208-216 base pairs 3' to the major RPO31 mRNA start sites. The RPO31 locus thus encodes the largest subunit of S. cerevisiae RNA polymerase III.