The human CD45 gene encodes five isoforms of a transmembrane tyrosine phosphatase that differ in their extracellular domains as a result of alternative splicing of exons 4 -6. Expression of the CD45 isoforms is tightly regulated in peripheral T cells such that resting cells predominantly express the larger CD45 isoforms, encoded by mRNAs containing two or three variable exons. In contrast, activated T cells express CD45 isoforms encoded by mRNAs lacking most or all of the variable exons. We have previously identified the sequences within CD45 variable exon 4 that control its level of inclusion into spliced mRNAs. Here we map the splicingregulatory sequences within CD45 variable exons 5 and 6. We show that, like exon 4, exons 5 and 6 each contain an exonic splicing silencer (ESS) and an exonic splicing enhancer (ESE), which together determine the level of exon inclusion in naïve cells. We further demonstrate that the primary activation-responsive silencing motif in exons 5 and 6 is homologous to that in exon 4 and, as in exon 4, binds specifically to the protein heterogeneous nuclear ribonucleoprotein L. Together these studies reveal common themes in the regulation of the CD45 variable exons and provide a mechanistic explanation for the observed physiological expression of CD45 isoforms.In order to attain proteomic and functional complexity, higher eukaryotes have developed numerous mechanisms to amplify the informational content of their relatively limited genomes. One such mechanism, alternative splicing, allows for the production of multiple unique mRNAs from a single gene through the differential inclusion of exons. Since the variant mRNAs produced by alternative splicing can each potentially encode for a functionally distinct protein, alternative splicing is recognized to be a ubiquitous and critical mechanism for regulating cellular function (1, 2).The importance of regulated alternative splicing in humans is exemplified by the CD45 gene, which encodes a transmembrane proteintyrosine phosphatase (3). CD45 phosphatase activity is critical for intracellular signaling in T cells in response to antigen stimulation, as indicated by the severe immunodeficiency observed in CD45-deficient mice and humans (4 -7). Five isoforms of the CD45 protein are expressed in humans as a result of alternative inclusion of exons 4 -6 (see Fig. 1A). In human T cells, the expression of the CD45 isoforms is tightly regulated throughout development and upon activation (3). In particular, naive peripheral T cells express significant amounts of the larger isoforms of CD45, which have high phosphatase activity and maintain the T cell receptor in a state primed for antigen recognition (8). Subsequent to antigen stimulation, however, there is a marked shift in the processing of CD45 pre-mRNA, such that the variable exons are predominantly excluded from the final message, resulting in expression of the smaller CD45 isoforms (9, 10). The smaller isoforms of CD45 are more prone to homodimerization than the larger isoforms (8, 11). Importantl...