Fractionation of an extract prepared from HeLa cells infected with vaccinia virus resulted in the separation of factors involved in vaccinia virus intermediate transcription. Two activities, VITF-A and VITF-B, in addition to the viral RNA polymerase are necessary and sufficient to direct intermediate transcription in vitro. VITF-B confers intermediate promoter specificity to an early-specific extract prepared from virus particles. A committed complex between VITF-B and the template can sequester VITF-A and RNA polymerase into a pre-initiation complex. VITF-B is further able to melt the promoter at the start site of transcription. Open complex formation is stimulated by ATP. In contrast to prokaryotic and eukaryotic pol III transcription, promoter melting is independent of the presence of RNA polymerase.