Earlier studies on genetic suppression ofprp24-1 byprp21-2 suggested an association between yeast Prp21 and Prp24 proteins, which are associated, respectively, with U2 snRNA and U6 snRNA. Here we report analyses of physical and functional interaction between these factors. Missense mutations in functionally important domains reside inprp21-2 andprp24-1. Two-hybrid assays do not detect interaction between wild-type or mutant proteins. Prp21-2 and Prp24-1 protein inprp21-2 orprp24-1 extracts can be heat-inactivatedin vitro. In contrast, heat-treated extracts from the revertant strainprp21-2 prp24-1 demonstrate allele-specific restoration of splicing. Suppression ofprp24-1 byprp21-2 does not cause coimmunoprecipitation of U2 and U6 snRNAs. We demonstrate the presence of Prp21 in the spliceosome assembly intermediate A2-1, and our data suggest the presence of Prp24 in the same complex. Kinetic analysis of assembly in heat-treated revertant extracts reveal a rate-limiting conversion of complex B to A2-1, suggesting transient association between the mutant proteins at this step. Our data also imply a requirement for Prp21 during B to A2-1 conversion. We conclude that a transient yet likely functional association between Prp21 and Prp24 occurs during spliceosome assembly.