The beta tropomyosin gene contains two internal exons which are spliced in a mutually exclusive manner. Exon 6B is specifically included in the mature transcripts expressed in skeletal muscle or cultured myotubes, while exon 6A is a myoblast- or smooth muscle-specific exon. The intron between them, which is never spliced in normal conditions, contains two characteristic features: first, the unusual location of the branch point at position -105 from the acceptor, and second, the presence of a very long pyrimidine stretch upstream of the skeletal muscle exon. In this study we designed a number of sequence modifications to investigate the role of these two elements and of a computer-predicted secondary structure in the mutually exclusive splicing of the two exons. We found that mutations in the skeletal exon as well as in the upstream intron could change in vivo the tissue-specific pattern as well as the mutually exclusive character of the two exons. Our results suggest that the unusual position of the branch point does not prevent the utilization of exon 6B in myoblasts and that the region around the acceptor site of exon 6B and the polypyrimidine tract have an important role in this control. Last, we discuss the possible implications of secondary structures.