We have separated and purified three factors from HeLa cell nuclear extracts that together can accurately cleave and polyadenylate pre-mRNAs containing the adenovirus L3 polyadenylation site. One of the factors is a poly(A) polymerase with a molecular weight of approximately 50-60 kd. The second activity is a cleavage factor with a native molecular weight in the range of 70-120 kd. The third component is a factor (cleavage and polyadenylation factor, CPF) that is needed for the cleavage reaction and, in addition, confers specificity to the poly(A) polymerase activity; the native molecular weight of CPF is approximately 200 kd. Poly(A) polymerase together with CPF is sufficient to specifically polyadenylate pre-mRNA substrates that have been precleaved at the poly(A) addition site. In contrast, all three components are required for accurate cleavage and polyadenylation of pre-mRNA substrates. Further purification of CPF by buoyant density centrifugation, ion exchange, and affinity column chromatography or by gel filtration demonstrates that CPF activity resides in a ribonucleoprotein and copurifies with U11 snRNP.