Adding NAD to murine T lymphocytes inhibits their functions and induces annexin V binding. This report shows that NAD induces cell death in a subset of T cells within seconds whereas others do not die until many hours later. Low NAD concentrations (<10 microM) suffice to trigger rapid cell death, which is associated with annexin V binding and membrane pore formation, is not blocked by the caspase inhibitor Z-VADfmk, and requires functional P2X7 receptors. The slower induction of death requires higher NAD concentrations (>100 microM), is blocked by caspase inhibitor Z-VADfmk, is associated with DNA fragmentation, and does not require P2X7 receptors. T cells degrade NAD to ADP-ribose (ADPR), and adding ADPR to T cells leads to slow but not rapid cell death. NAD but not ADPR provides the substrate for ADP-ribosyltransferase (ART-2)-mediated attachment of ADP-ribosyl groups to cell surface proteins; expression of ART-2 is required for NAD to trigger rapid but not slow cell death. These results support the hypothesis that cell surface ART-2 uses NAD but not ADPR to attach ADP-ribosyl groups to the cell surface, and that these groups act as ligands for P2X7 receptors that then induce rapid cell death. Adding either NAD or ADPR also triggers a different set of mechanisms, not requiring ART-2 or P2X7 receptors that more slowly induce cell death.