Splicing of SV40 early pre-mRNA to alternative large-T and small-t mRNAs involves the utilization of multiple lariat branch sites. To determine the functional significance of these sites, we constructed and analyzed a set of base substitution mutants in which the major branch acceptors were altered, either singly or in combination. The ratio of large-T to small-t mRNAs produced in vivo was found to vary by over 100-fold between different mutants. When splicing was assayed in vitro with a standard pre-RNA, which results in splicing almost exclusively to large-T mRNA, the patterns of branch site utilization were altered dramatically, although the mutations were essentially without effect on splicing efficiency. However, use of a 5' truncated pre-RNA, which results in a splicing pattern that reflects the in vivo alternative splicing potential of this pre-RNA, revealed a strong correlation between the effects of the base substitutions on branch site selection and alternative splice-site utilization. An RNase protection analysis of factor interactions with the 5' splice sites and branch sites in wild-type and mutant pre-RNAs suggests that a competition for different branch sites plays a crucial role in the assembly of 'alternative' spliceosomes, thereby controlling alternative splice-site selection.