Specific antibodies directed against each polypeptide component of yeast RNA polymerases A or B were prepared and their affinity spectrum determined by protein blot immunodetection. The majority of enzyme A or B subunits were specifically recognized by their respective antiserum. A direct correspondence was established between the polypeptides immunologically related in the three forms of RNA polymerases A, B, and C by reacting the different antibodies with enzymes subunits transferred to a nitrocellulose membrane. Subunit-specific antibodies and antibodies to native enzymes A and B were used to probe the activity of RNA polymerases A, B, and C. Based on DNA protection experiments, the largest subunit of enzymes A and B as well as the common subunit ABC23 appear to be involved in DNA binding.