9G8 protein belongs to the conserved serine/arginine-rich (SR) protein family, whose members exhibit multiple functions in constitutive and alternative splicing. We have previously shown that 9G8 primary transcripts are subjected to alternative splicing by excision/retention of intron 3 and to a tissue specific modulation. Because both 5'- and 3'-splice sites of intron 3 appear to be suboptimal in vertebrates, we tested the 9G8 intron 3 as a novel model system of alternative splicing. By using an in vitro approach and a mutational analysis, we have identified two purine-rich exonic splicing enhancers (ESE) located in exon 4 and a (GAA)(3) enhancer located in exon 3. These elements act in concert to promote efficient splicing activation both in vitro and in vivo. Titration experiments with an excess of exonic enhancers or SR-specific RNA targets strongly suggest that SR proteins are specifically involved in the activation process. Although ASF/SF2 was expected to interact the most efficiently with ESE according to the enhancer sequences, UV cross-linking coupled or not to immunopurification demonstrates that 9G8 is highly recruited by the three ESE, followed by SC35. In contrast, ASF/SF2 only binds significantly to the (GAA)(3) motif. S100 complementation experiments with individual SR proteins demonstrate that only 9G8 is able to fully restore splicing of intron 3. These results, and the fact that the exon 3 and 4 ESE sequences are conserved in vertebrates, strongly suggest that the alternative splicing of intron 3 represents an important step in the regulation of the expression of 9G8.