Abstract PRP8 encodes a highly conserved U5 snRNP protein required for spliceosome assembly and later steps of pre-mRNA splicing. We recently identified a novel allele, prp8-101, that specifically impairs recognition of the undine tract that precedes most yeast 3′ slice sites. We carried out extensive mutagenesis of the gene and selected for new alleles that confer a phenotype similar to that of prp8-101. The strongest alleles cause changes in one of two amino acids in the C-terminal portion of the protein. We also identified a second class of PRP8 mutant that affects the fidelity of 3′ splice site utilization. These alleles suppress point mutations in the PyAG motif at the 3′ splice site and do not alter uridine tract recognition. The strongest of these alleles map to a region directly upstream of the pp8-101-like mutations. These new PRP8 alleles define two separable functions of Prp8p, required for specificity of 3′ splice site selection and fidelity of 3′ splice site utilization, respectively. Taken together with other recent biochemical and genetic data, our results suggest that Prp8p plays a functional role at the active site of the spliceosome during the second catalytic step of splicing.