We describe a new approach to elucidate the role of 3'-end processing in pre-mRNA splicing in vivo using the influenza virus NS1A protein. The effector domain of the NS1A protein, which inhibits the function of the CPSF and PABII factors of the cellular 3'-end-processing machinery, is sufficient for the inhibition of not only 3'-end formation but also the splicing of single-intron pre-mRNAs in vivo. We demonstrate that inhibition of the splicing of single-intron pre-mRNAs results from inhibition of 3'-end processing, thereby establishing that 3'-end processing is required for the splicing of a 3' terminal intron in vivo. Because the NS1A protein causes a global suppression of 3'-end processing in trans, we avoid the ambiguities caused by the activation of cryptic poly(A) sites that occurs when mutations are introduced into the AAUAAA sequence in the pre-mRNA. In addition, this strategy enabled us to establish that the function of a particular 3'-end-processing factor, namely CPSF, is required for the splicing of single-intron pre-mRNAs in vivo: splicing is inhibited only when the effector domain of the NS1A protein binds and inhibits the function of the 30-kDa CPSF protein in 3'-end formation. In contrast, the 3'-end processing factor PABII is not required for splicing. We discuss the implications of these results for cellular and influenza viral mRNA splicing.