SV40 early pre-mRNA is alternatively spliced to produce large T and small t mRNAs by use of different 5'-splice sites and a shared 3'-splice site. The large T splicing pathway uses multiple lariat branch sites, whereas small t splicing, constrained by its small intron size, can use only one. We exploited this situation to test the hypothesis that RNA-RNA base pairing between U2 snRNA and the branch site sequence is important in mammalian pre-mRNA splicing by constructing and analyzing several mutations in the small t pre-mRNA branch site (UUCUAAU). All of the mutations resulted in substantial decreases in small t splicing relative to large T. To test whether these effects resulted from decreased base pairing with U2 snRNA, compensatory mutations were introduced at the appropriate positions (nucleotides 34-36) in a cloned human U2 gene. All branch site mutations tested (four separate single base substitutions and two triple mutations) were suppressed (i.e., small t splicing was increased) by the appropriate U2 mutations. These results establish that recognition of the poorly conserved mammalian pre-mRNA branch site sequence by U2 snRNP can involve base-pairing.