The gene product of the vaccinia virus open reading frame D6R was synthesized in bacteria and used to raise antiserum against the protein. Using the antiserum as a probe, we demonstrated that the D6R protein is a component of the virion particle, localized to the virus core structure. The D6R protein, purified from virions, has been shown to copurify with the vaccinia virus early transcription factor (VETF). The apparent molecular weight of the D6R polypeptide is identical to that of the smaller of the two VETF-associated polypeptides. Antibodies directed against D6R block both the early promoter-binding and DNA-dependent ATPase activities of VETF, supporting the identity of D6R as a VETF-associated polypeptide. An ATP-binding site was inferred near the amino terminus of the derived D6R amino acid sequence. Thus, the D6R polypeptide could be the source of the ATPase activity associated with VETF. The D6R gene was shown previously to belong to the late class of vaccinia virus genes. Synthesis of the VETF at late times after infection suggests a cascade model for vaccinia virus gene regulation in which class-specific transcription factors are synthesized at the previous phase of the infectious cycle.