What determines the precise sites of cleavage in the two transesterification reactions of messenger RNA (mRNA) splicing is a major unsolved question.Mutation of the invariant G (guanosine) at position 5 of 5"prime" splice sites in Saccharomyces cerevisiae introns activates cleavage at nearby aberrant sites. A genetic approach was used to test the hypothesis that a base-pairing interaction between the 5"prime" splice site and the invariant ACAGAG sequence of U6 is a determinant of 5"prime" splice site choice. Mutations in U6 or the intron (or both) that were predicted to stabilize the interaction suppressed aberrant cleavage and increased normal cleavage. In addition, a mutation in the ACAGAG sequence suppressed mutations of the 3"prime" splice site dinucleotide. These data can fit a model for the spliceosomal active site comprised of a set of RNA-RNA interactions between the intron, U2 and U6.