Splicing of the last intron (intron D) of the bovine growth hormone pre-mRNA requires the presence of a downstream exonic splicing enhancer (ESE). This enhancer is contained within a 115-nucleotide FspI-PvuII (FP) fragment located in the middle of the last exon (exon 5). Previous work showed that the splicing factor SF2/ASF binds to this FP region and stimulates splicing of intron D in vitro. However, the precise sequences recognized by SF2/ASF within the FP region had not been determined. Here we used multiple strategies to map the SF2/ASF binding sites and determine their importance for ESE function. Taking advantage of the fact that SF2/ASF ultraviolet (UV) cross-links specifically to RNA containing the FP sequence, we first mapped a major SF2/ASF binding site by UV cross-linking and reverse transcription. This strategy identified a 29-nucleotide SF2/ASF binding region in the middle of the FP sequence containing the 7-nucleotide purine-rich motif described previously. Interestingly, this binding region is neither sufficient, nor absolutely required for SF2/ASF-mediated splicing, suggesting that additional SF2/ASF binding sites are present. The location of these additional sites was determined by electrophoretic mobility shift analysis of various subfragments of the FP sequence. Antisense 2'-O-methyl oligoribonucleotides complementary to selected SF2/ASF binding sites block bovine growth hormone intron D splicing. Thus, multiple SF2/ASF binding sites within the exonic splicing enhancer contribute to maximal enhancer activity.