We have resolved the in vitro late transcription system of vaccinia virus into four components consisting of RNA polymerase and three accessory factors. One of these additional factors is a 30-kDa protein which was previously shown to be required for late transcription in vitro and was indirectly shown to be the product of the G8R open reading frame. Another factor, of 17 kDa, was previously identified as a possible late transcription factor by an assay which demonstrated that the gene encoding it, A1L, was required for late gene expression in vivo. The G8R and A1L open reading frames have now been cloned into a baculovirus expression system, and the corresponding proteins have been purified. Both are necessary for late transcription in vitro, confirming that these intermediate genes encode late transcription factors. The third factor has a sedimentation coefficient consistent with a protein of 32 to 38 kDa. Experimental results suggest that this is a previously unidentified factor encoded by a vaccinia virus early gene. The RNA polymerase functioning in this system was purified from vaccinia virus-infected cells; however, it can be complemented by the RNA polymerase which is packaged in virions. The three smaller proteins and RNA polymerase are all necessary, and together are sufficient, for the synthesis of late viral mRNA in vitro.