Human CD100, the first semaphorin identified in the immune system, is a transmembrane protein involved in T-cell activation. In the present study, we showed that activation of peripheral blood or tonsillar B lymphocytes induced the expression of CD100 in CD38⁺CD138⁻ cell populations, including in CD148⁺ subpopulations, thus expressing a memory B-cell–like phenotype. Using an in vitro enzymatic assay, we found that protein tyrosine phosphatase (PTP) activities were immunoprecipitated with CD100 in these cell populations, which were isolated by cell sorting, as well as in most B-cell lines representing various stages of B-cell differentiation. Immunodepletion and Western blotting experiments demonstrated that CD45 was the PTP associated with CD100 in cell lines displaying pre-B, activated B, and pre-plasma cell phenotypes. CD45 also accounted for PTP activity immunoprecipitated with CD100 in CD38⁺CD138⁻ cells sorted after activation of peripheral blood or tonsillar B lymphocytes. In contrast, no CD100-CD45 association was observed in plasma cell lines corresponding to the terminal B-cell differentiation stage. CD148, the other transmembrane PTP known to be implicated in lymphocyte signaling pathways, was either only partly involved in the CD100-associated PTP activity or not expressed in plasma cell lines, indicating the association of CD100 with another main PTP. Our data show that CD100 is differentially expressed and can functionally associate with distinct PTPs in B cells depending on their activation and maturation state. They also provide evidence for a switch in the CD100-associated PTP at terminal stage of B-cell differentiation.