Partially purified DNA-dependent RNA polymerase from infectious vaccinia virus particles exhibits the following two activities: 1) specific transcription of double-stranded DNA templates containing vaccinia early promoters and 2) nonspecific transcription of single-stranded DNA templates. After further purification of the RNA polymerase, specific transcriptase activity was selectively diminished suggesting the loss of a transcription factor. In agreement with the latter hypothesis, transcriptase activity could be reconstituted by mixing the purified RNA polymerase with certain column fractions. A quantitative complementation assay was developed and used to locate the transcription factor during successive column chromatography steps. The factor eluted as a single peak of activity from single strand DNA-cellulose and phosphocellulose columns. An observation that the transcription factor binds specifically to vaccinia early promoter sequences was exploited in the final affinity chromatography steps. The purified factor was separated from all previously identified vaccinia enzymes and contained two polypeptides of Mr 77,000 and 82,000. A DNA-dependent ATPase activity also copurified with the transcription factor. Although a single template was used for assays during isolation, the purified factor stimulated transcription of three other early genes by 20-30-fold suggesting that it has a general role in conferring promoter specificity for initiation of early transcription.