Genes encoding yeast RNA polymerase II subunits were cloned. Efficient isolation of these genes was accomplished by probing a phage lambda gt11 recombinant DNA expression library with polyvalent antibodies directed against purified yeast RNA polymerase II. The identity of genes that specify the largest RNA polymerase II subunits, the 220,000- and 150,000-dalton polypeptides, was confirmed by competitive radioimmune assay. Both of these genes exist in single copy in the yeast Saccharomyces cerevisiae.