Trans-splicing requires that 5′ and 3′ splice sites be independently recognized. Here, we have used mutational analyses and a sensitive nuclease protection assay to determine the mechanism of trans-3′ splice site recognition in vitro. Efficient recognition of the 3′ splice site is dependent upon both the sequence of the 3′ splice site itself and enhancer elements located in the 3′ exon. We show that the presence of three distinct classes of enhancers results in increased binding of U2 snRNP to the branchpoint region. Several lines of evidence strongly suggest that the increased binding of U2 snRNP is mediated by U2AF. These results expand the roles of enhancers in constitutive splicing and provide direct support for the recruitment model of enhancer function.