Purine-rich sequences within exons are proposed to promote proper splicing as splicing enhancers. In order to test this supposition in the dystrophin gene, which is characterized by the large size of its introns, purine-rich sequences from three exons (exons 43, 46 and 53) were examined for their splicing enhancer activity by using a Drosophila doublesex pre-mRNA. The most powerful activating effect on upstream intron splicing was seen with a sequence from exon 43. A sequence from exon 53 showed relatively low activity, whilst that from exon 46 had little effect. To characterize the splicing enhancer sequences in exons 53 and 46 further, entire exons were divided into 30nt fragments that were examined separately for their splicing enhancer activity. In exon 53, two fragments located at the 5' and 3' ends, respectively, had strong splicing enhancer activity, although they were not the most purine-rich regions of the exon. In contrast, all of the fragments derived from exon 46 had little activity. These results suggest that different exons in the dystrophin transcript are subject to different mechanisms of control by the splicing machinery.