The association of a 94,000-Da viral polypeptide, called Rap94, with 30-40% of the multisubunit DNA-dependent RNA polymerase molecules purified from infectious vaccinia virus particles was established by immunoaffinity chromatography. The submolar amount of Rap94, relative to RNA polymerase, was confirmed by quantitative immunoblotting of total virion extracts. Only the RNA polymerase molecules containing Rap94 could functionally interact with VETF, the vaccinia virus early transcription factor, to transcribe a double-stranded DNA template regulated by a viral early stage promoter. Rap94 was required for the synthesis of short oligoribonucleotides and for the formation of stable ternary transcription complexes. With a nonspecific single-stranded DNA template, however, the Rap94-deficient polymerase had greater catalytic activity than the Rap94-containing polymerase. These data support a model in which Rap94 confers specificity to the RNA polymerase for promoters of early stage genes.