Alternative splicing of vertebrate beta-tropomyosin transcripts ensures mutually exclusive expression of internal exons 6A and 6B in nonmuscle and skeletal muscle cells, respectively. Recently, we reported that this splicing regulation requires species-specific elements, since the splicing profile for the chicken, rat, and Xenopus beta-tropomyosin alternative exons is not reproduced in transfection experiments when heterologous myogenic cells are used. By analyzing the splicing pattern of hybrid chicken/rat beta-TM constructions transfected into both quail and mouse cell lines, we demonstrate that chicken beta-tropomyosin exon 6A is flanked by stronger splicing signals than rat exon 6A, thus leading to the misregulation of splicing in heterologous cells. We have characterized three splicing signals that contribute to this difference: 1) nonconsensus nucleotide differences at positions +4 and +6 in the donor site downstream of exon 6A, 2) differences in the pyrimidine composition between the branch site and acceptor site upstream of exon 6A, and 3) a pyrimidine-rich intronic exon 6A splicing enhancer present upstream of exon 6A only in the chicken beta-TM gene. The functional divergence between splicing signals in two homologous vertebrate genes reveals species-specific strategies for proper modulation of splicing of alternative exons.