It has previously been shown in vivo that bovine papillomavirus represses its late gene expression via a 5′ splice site sequence located upstream of the late polyadenylation signal. Here, the mechanism of repression is determined by in vitro analysis. U1 snRNP binding to the 5′ splice site results in inhibition of polyadenylation via a direct interaction with poly(A) polymerase (PAP). Although the inhibitory mechanism is similar to that used in U1A autoregulation, U1A within the U1 snRNP does not contribute to PAP inhibition. Instead the U1 70K protein, when bound to U1 snRNA, both interacts with and inhibits PAP. Conservation of the U1 70K inhibitory domains suggests that polyadenylation regulation via PAP inhibition may be more widespread than previously thought.