mRNA made in eukaryotic cells typically has a 3' poly(A) tail that is added posttranscriptionally. To investigate mechanisms by which 3' poly(A) is formed, we identified the genes for the two vaccina virus-encoded polypeptides, VP55 and VP39. Primer-dependent polyadenylation activity was associated exclusively with purified VP55-VP39 heterodimer, which, although stable to column chromatography and glycerol gradient sedimentation, was readily dissociated by antibody to an N-terminal peptide of VP55. Poly(A) polymerase activity was associated with immunopurified VP55, but not with immunopurified or chromatographically purified VP39. VP39 was, however, required for the formation of long poly(A) molecules, in conjunction with either purified VP55 or low concentrations of the heterodimer, and was shown to bind free poly(A). Thus, a catalytic polypeptide and a dissociable poly(A)-binding stimulatory factor each contribute to poly(A) tail formation. No prokaryotic or eukaryotic homologs of either polypeptide were detected in sequence data bases, consistent with the absence of previously reported poly(A) polymerase genes from any source.