We have investigated how the upstream sequence element (USE) of the lamin B2 poly(A) signal mediates efficient 3'-end formation. In vitro analysis demonstrates that this USE increases both the efficiency of 3'-end cleavage and the processivity of poly(A) addition. Cross-linking using selectively labeled synthetic RNAs confirms that cleavage stimulation factor interacts with the sequences downstream of the cleavage site, while electrophoresis mobility shift assays demonstrate that the USE directly stabilizes the binding of the cleavage and polyadenylation specificity factor to the poly(A) signal. Thus in common with other poly(A) signals, the lamin B2 USE directly enhances the binding of basal poly(A) factors. In addition, a novel 55-kDa protein binds to the USE and the core poly(A) signal and appears to inhibit cleavage. The binding activity of this factor appears to change during the cell cycle, being greatest in S phase, when the lamin B2 gene is transcribed.