The rat β-tropomyosin gene encodes two isoforms, termed skeletal muscle β-tropomyosin and fibroblast plast tropomyosin 1 (TM-1), via an alternative RNA processing mechanism. The gene contains 11 exons. Exons 1-5 and exons 8 and 9 are common to all mRNAs expressed from the gene. Exons 6 and 11 are used in fibroblasts, as well as smooth muscle , whereas exons 7 and 10 are used only in skeletal muscle. In the present studies we focused on the mutually exclusive internal alternative splice choice involving exon 6 (fibroblast-type splice) and exon 7 (skeletal muscle-type splice). We have indentified two distinct elements in the intron, upstream of exon 7, involved in splice site selection. The first element is comprised of a polypyrimidine tract located 89-143 nucleotides upstream of the 3 splice site, which specifies the location of the lariat branchpoints used, 144-153 nucleotides upstream of exon 7. The 3 splice site AG dinucleotide has no role in selection of these branchpoints. The second element is comprised of intron sequences located between the polypyrimidine tract and the 3 splice site of exon 7. It contains an important determinant in alternative splice site selection, because deletion of these sequences results in the use of the skeletal muscle-specific exon in nonmuscle cells. We propose that the use of lariat branchpoints located far upstream from 3 splice site may be a general feature of some alternatively excised introns, reflecting the presence of regulatory sequences located between the lariat branch site and the 3 splice site. The data also indicate that alternative splicing of the rat β-tropomyosin gene is regulated by a somewhat different mechanism from that described for rat α-tropomyosin gene and the transformer-2 gene of Drosophila melanogaster