The mRNAs encoding the vaccinia virus F17 protein and the cowpox A-type inclusion protein are known to possess sequence-homogeneous 3' ends, generated by a post-transcriptional cleavage event. By using partially purified extracts, we have previously shown that the same factor probably cleaves both the F17 and A-type inclusion protein transcripts and that the cleavage factor is either virus-coded or virus-induced during the post-replicative phase of virus replication. In this study, we have purified the cleavage factor from vaccinia-infected HeLa cells using column chromatography and gel filtration. The factor eluted from the gel filtration column with an apparent molecular mass of approximately 440 kDa. Mass spectrometric analyses of the proteins present in the peak active fractions revealed the presence of at least one vaccinia protein with a high degree of certainty, the H5R gene product. To extend this finding, extracts were prepared from HeLa cells infected with vaccinia virus overexpressing His-tagged H5, chromatographed on a nickel affinity column, and eluted using an imidazole gradient. Cleavage activity eluted with the peak of His-tagged H5. Gel filtration of the affinity-purified material further demonstrated that cleavage activity and His-tagged H5 co-chromatographed with an apparent molecular mass of 463 kDa. We therefore conclude that H5 is specifically associated with post-transcriptional cleavage of F17R transcripts. In addition, we show that dephosphorylation of a cleavage competent extract with a nonspecific phosphatase abolishes cleavage activity implying a role for phosphorylation in cleavage activity.