The YAG/ consensus sequence at the 3′ end of introns (the slash indicates the location of the 3′ splice site) is essential for catalysis of the second step of pre-mRNA splicing. Little is known about the interactions formed by these three nucleotides in the spliceosome. Although previous observations have suggested that the G of the YAG/ interacts with the first nucleotide of the /GUA consensus sequence at the 5′ end of the intron, additional interactions have not been identified. Here we report several striking genetic interactions between A+3 of the 5′ /GUA with Y−3 of the 3′ YAG/ and G50 of the highly conserved ACAGAG motif in U6 snRNA. Two mutations in U6 G50 of the ACAGAG can weakly suppress two mutations in A+3 of the 5′ /GUA. This suppression is significantly enhanced upon the inclusion of a specific mutation Y−3 in the 3′ YAG/. RNA analysis confirmed that the severe splicing defect observed in A+3 and Y−3 double mutants can be rescued to near wild-type levels by the mutations in U6 G50. The contributions of each mutation to the genetic interaction and the strong position specificity of suppression, combined with previous findings, support a model in which the 5′ /GUA and the GAG of U6 function in binding the 3′ YAG/ during the second catalytic step.