Analysis of the in vitro splicing products of RNA precursors containing tandem duplications of the 5' or 3' splice sites of human beta-globin IVS 1 revealed that exon sequences play an important role in the relative use of the duplicated sites. These studies also show that the proximity of the 5' and 3' splice sites is an important determinant in the selection of splice-sites. Deletion, substitution, or even subtle changes of exon sequences can alter the pattern of splice-site selection, and in many cases the splice site adjacent to the altered exon is not used. The relative use of the duplicated splice sites can also be altered by diluting the splicing extract, suggesting that factors involved in splice-site selection are limiting.