Extracellular NAD(+) and ATP trigger the shedding of CD62L and the externalization of phosphatidylserine on murine T cells. These events depend on the P2X(7) ion channel. Although ATP acts as a soluble ligand to activate P2X(7), gating of P2X(7) by NAD(+) requires ecto-ADP-ribosyltransferase ART2.2-catalyzed transfer of the ADP-ribose moiety from NAD(+) onto Arg125 of P2X(7). Steady-state concentrations of NAD(+) and ATP in extracellular compartments are highly regulated and usually are well below the threshold required for activating P2X(7). The goal of this study was to identify possible endogenous sources of these nucleotides. We show that lysis of erythrocytes releases sufficient levels of NAD(+) and ATP to induce activation of P2X(7). Dilution of erythrocyte lysates or incubation of lysates at 37 degrees C revealed that signaling by ATP fades more rapidly than that by NAD(+). We further show that the routine preparation of primary lymph node and spleen cells induces the release of NAD(+) in sufficient concentrations for ART2.2 to ADP-ribosylate P2X(7), even at 4 degrees C. Gating of P2X(7) occurs when T cells are returned to 37 degrees C, rapidly inducing CD62L-shedding and PS-externalization by a substantial fraction of the cells. The "spontaneous" activation of P2X(7) during preparation of primary T cells could be prevented by i.v. injection of either the surrogate ART substrate etheno-NAD or ART2.2-inhibitory single domain Abs 10 min before sacrificing mice.