CD100 is a 150-kDa human surface glycoprotein implicated in T cell activation. Using BB18 and BD16 mAbs to two discrete epitopes of the CD100 molecule, we have shown previously that triggering the CD100 molecule through the BB18 epitope is comitogenic with PMA, whereas it lacks a detectable effect on CD2- and CD3-induced PBMC proliferation. Conversely, triggering the CD100 molecule through the BD16-defined epitope only exerts an effect on CD2- and CD3-induced PBMC proliferation. In the present study, we investigated the molecular relationship between CD100 and another surface structure involved in human T cell proliferation, the CD45 phosphatase. We show in this study that CD100 is associated with CD45 and that this association has functional significance. The association was demonstrated using coimmunoprecipitation and detection of CD45 enzymatic PTPase activity. Furthermore, we show that the association is increased during T cell activation and that triggering CD45 molecules through discrete epitopes induces the down-modulation of CD100 molecules at the cell surface. Interestingly, we demonstrate that this modulation could be attributed to the shedding of a soluble form of CD100 in the culture supernatant. Finally, one of the functional consequences of this T cell activation-induced CD100-CD45 association is revealed by the finding that CD100 mAbs have an effect on CD45-induced T cell aggregation.