Six different protein factors are required for the specific cleavage and polyadenylation of pre-mRNA in mammals. Whereas four of them have been purified and most of their components cloned, cleavage factor Im (CF Im) and cleavage factor IIm (CF IIm) remained poorly characterized. We report here the separation of CF Im from CF 11m and the purification of CF Im to near homogeneity. Three polypeptides of 68, 59, and 25 kDa copurify with CF Im activity. All three polypeptides can be UV cross-linked to a cleavage and polyadenylation substrate in the presence of a large excess of unspecific competitor RNA, but not to a splicing-only substrate. No additional protein factor is required for the binding of CF Im to pre-mRNA. Gel retardation experiments confirmed the results obtained by UV cross-linking. In addition, we could show that CF Im stabilizes the binding of the cleavage and polyadenylation specificity factor (CPSF) to pre-mRNA and that CPSF and CF Im together form a slower migrating complex with pre-mRNA than the single protein factors. Cleavage stimulation factor (CstF) and poly(A) polymerase (PAP) had no detectable effect on the binding of CF Im to pre-mRNA. Furthermore, the CstF-CPSF-RNA as well as the CstF-CPSF-PAP-RNA complex are supershifted and stabilized upon the addition of CF Im.