Previous studies have identified a single nucleotide polymorphism that significantly increases the splicing of variable exon 4 in transcripts of the human protein-tyrosine phosphatase CD45. Strikingly, the presence of this polymorphism correlates with susceptibility to the autoimmune disease multiple sclerosis. In this study we investigated the mechanism by which the polymorphism enhances splicing of CD45 exon 4. We found that at least four distinct splicing regulatory elements exist within exon 4 and that the strongest of these elements is an exonic splicing silencer (designated ESS1), which is disrupted by the polymorphism. We show that ESS1 normally functions to repress the weak 5' splice site (ss) of CD45 exon 4. The ESS1 sequence also suppresses the splicing of a heterologous 5'ss and associates with a specific complex in nuclear extracts. We further demonstrate that ESS1 is juxtaposed to a purine-rich enhancer sequence that activates the use of the 5'ss of exon 4. Thus, proper functioning of the immune system is dependent on a complex interplay of regulatory activities that mediate the appropriate splicing of CD45 exon 4.