Activation of the damage-associated molecular pattern receptor P2X7 by extracellular ATP induces the shedding of cell surface molecules, including CD23, from leukocytes. The mechanisms involved in this process however remain poorly understood. Using a flow cytometric assay, we demonstrated that ATP-induces the rapid shedding of CD23 from the surface of human multiple myeloma RPMI 8226 and peripheral blood B cells with a t1/2 of 7 min. The P2X7 antagonist, AZ10606120, impaired ATP-induced CD23 shedding by 89%. Moreover, P2X7-induced CD23 shedding was impaired by a broad-spectrum metalloprotease inhibitor, BB-94, by 63% and the ADAM10 inhibitor, GI254023X, by 67%. RT-PCR confirmed the expression of ADAM10 in B cells. P2X7-induced CD23 shedding was not dependent on changes in extracellular Na+, K+ and Ca2+ concentrations, and was unaffected by a panel of enzyme inhibitors targeting various kinases and phospholipases previously implicated in P2X7-mediated signaling events. ATP also induced the rapid shedding of the ADAM10 substrate, CXCL16, with a t1/2 of 1 min. AZ10606120 impaired ATP-induced CXCL16 shedding by 86%. Moreover, P2X7-induced CXCL16 shedding was impaired by BB-94 by 77% and GI254023X by 87%. This data indicates that human P2X7 activation induces the rapid shedding of CD23 and CXCL16, and that this process involves ADAM10. Thus, P2X7 may represent a novel target in disorders involving CD23 or CXCL16.