Alternative splicing of chicken beta-tropomyosin (beta-TM) pre-mRNAs ensures that in nonmuscle cells, only exon 6A is expressed, whereas in skeletal muscle, exon 6B is utilized preferentially. We have previously shown that efficient splicing of the nonmuscle exon 6A requires two pyrimidine-rich splicing enhancers (S4 and I5Y) that are present in the introns flanking exon 6A. Here, we examined the function of the S4 and I5Y elements by replacing them within beta-TM minigenes by other pyrimidine- and purine-rich sequence elements and analyzing splicing in transfected quail nonmuscle and muscle cells. Several features of these splicing regulatory elements were revealed by this study. First, a wide variety of pyrimidine-rich sequences can replace the intronic S4 splicing enhancer, indicating that pyrimidine composition, rather than sequence specificity, determines activity for this element. Second, one type of purine-rich sequence (GARn), normally found within exons, can also replace the S4 splicing enhancer. Third, the diverse elements tested exhibit differential activation of the splice sites flanking exon 6A and different positional constraints. Fourth, the strength of the S4 splicing enhancer is appropriately set to obtain proper regulation of the transition from exon 6A splicing in myoblasts to exon 6B splicing in myotubes, but this splicing regulatory element is not the target for cell-type-specific splicing factors.