The gene, rpo 132, encoding the second-largest subunit of the vaccinia virus DNA-dependent RNA polymerase was identified and sequenced. Two complementary approaches, involving antiserum to purified vaccinia virus RNA polymerase, were used to locate the rpo 132 gene. One method involved the screening of a lambda gt11 library of vaccinia virus genome fragments and the other was based on the immunoprecipitation and polyacrylamide gel electrophoresis of the in vitro translation products of mRNA that hybridized to immobilized vaccinia virus DNA. The deduced open reading frame of the rpo 132 gene predicted a polypeptide of 1164 amino acid residues with sequence similarities to the second-largest RNA polymerase subunits of eubacteria, archaebacteria, and eukaryotes as well as to other poxviruses. Transcriptional analyses indicated that rpo 132 has both early and late RNA start sites and is expressed throughout infection.