Yeast RNA polymerases A and C share an approximately equal to 40 kd subunit. We have identified, sequenced, and mutagenized in vitro the AC40 subunit gene. The RPC40 gene is unique in the yeast genome and is required for cell viability. This gene contains an open reading frame encoding a 37.6 kd protein having no significant homology with bacterial RNA polymerase subunits. The promoter region contains a 19 bp sequence also present in the largest subunit of RNA polymerase C. It also contains a well-conserved RPG box, a sequence found in the promoter region of many genes encoding the translational apparatus. A novel, plasmid-shuffling method was developed to isolate a large number of RPC40 ts mutants. One of these, ts4, was shown to be defective in the synthesis of RNA polymerases A and C at the restrictive temperature. In contrast, RNA polymerase B was made normally.