IVS1, an intron derived from the rat fibronectin gene, is spliced inefficiently in vitro, involving the use of three alternative branch sites. Mutation of one branch point site, BP3, so as to increase complementarity to U2 snRNA resulted in exclusive use of that site and improved splicing efficiency, indicating that the wild type BP3 site is one determinant of poor IVS1 splicing. Deletions within the polypyrimidine tract had a variable effect on splicing efficiency and altered the pattern of branch site usage. Selection of each branch site was influenced negatively by purine substitutions ca. 20 nucleotides downstream. It is proposed that all three IVS1 branch sites are pyrimidine tract-dependent. Pyrimidine tract deletions also influenced the crosslinking of PTB (the polypyrimidine tract-binding protein), hnRNP C, and splicing factor U2AF65. All three proteins bound preferentially to distinct regions within the polypyrimidine tract and thus are candidates for mediating pyrimidine tract-dependent branch site selection. The findings indicate the complexity of the IVS1 polypyrimidine tract and suggest a crucial role for this region in modulating branch site selection and IVS1 splicing.