The choice of a 3' splice site in Saccharomyces cerevisiae introns involves recognition of a uridine-rich tract upstream of the AG dinucleotide splice junction. By isolating mutants that eliminate the normal preference for uridine-containing 3' splice sites in a cis-competition, we identified a mutation that is an allele of PRP8, prp8-101. This was unexpected because previous analysis has demonstrated that the U5 snRNP protein encoded by PRP8 is required for spliceosome assembly prior to the first catalytic step of splicing. In contrast, the uridine recognition defect caused by the prp8-101 mutation selectively inhibits the second catalytic step of splicing. This defect is seen not only in 3' splice site cis-competitions but also in the splicing of an unusual intron in the TUB3 gene and in the ACT1 intron when utilization of its 3' splice site is rate limiting for splicing. Consistent with a direct role in 3' splice site selection, Prp8 can be cross-linked to the 3' splice site during the splicing reaction. These data demonstrate a novel function for Prp8 in 3' splice site recognition and utilization.