The human IGF-I gene has six exons, four of which are alternatively spliced. Variations in splicing involving exon 5 may occur, depending on the tissue type and hormonal environment. To study the regulation of splicing to IGF-I exon 5, we established an in vitro splicing assay, using a model pre-mRNA containing IGF-I exons 4 and 5 and part of the intervening intron. Using a series of deletion mutants, we identified an 18-nucleotide purine-rich splicing enhancer in exon 5 that increases the splicing efficiency of the upstream intron from 6 to 35%. We show that the serine-arginine protein splicing factor-2/alternative splicing factor specifically promotes splicing in cultured cells and in vitro and is recruited to the spliceosome in an enhancer-specific manner. Our findings are consistent with a role for splicing factor-2/alternative splicing factor in the regulation of splicing of IGF-I alternative exon 5 via a purine-rich exonic splicing enhancer.