Micromolar concentrations of extracellular beta-NAD(+) (NAD(e)(+)) activate human granulocytes (superoxide and NO generation and chemotaxis) by triggering: (i) overproduction of cAMP, (ii) activation of protein kinase A, (iii) stimulation of ADP-ribosyl cyclase and overproduction of cyclic ADP-ribose (cADPR), a universal Ca(2+) mobilizer, and (iv) influx of extracellular Ca(2+). Here we demonstrate that exposure of granulocytes to millimolar rather than to micromolar NAD(e)(+) generates both inositol 1,4,5-trisphosphate (IP(3)) and cAMP, with a two-step elevation of intracellular calcium levels ([Ca(2+)](i)): a rapid, IP(3)-mediated Ca(2+) release, followed by a sustained influx of extracellular Ca(2+) mediated by cADPR. Suramin, an inhibitor of P2Y receptors, abrogated NAD(e)(+)-induced intracellular increases of IP(3), cAMP, cADPR, and [Ca(2+)](i), suggesting a role for a P2Y receptor coupled to both phospholipase C and adenylyl cyclase. The P2Y(11) receptor is the only known member of the P2Y receptor subfamily coupled to both phospholipase C and adenylyl cyclase. Therefore, we performed experiments on hP2Y(11)-transfected 1321N1 astrocytoma cells: micromolar NAD(e)(+) promoted a two-step elevation of the [Ca(2+)](i) due to the enhanced intracellular production of IP(3), cAMP, and cADPR in 1321N1-hP2Y(11) but not in untransfected 1321N1 cells. In human granulocytes NF157, a selective and potent inhibitor of P2Y(11), and the down-regulation of P2Y(11) expression by short interference RNA prevented NAD(e)(+)-induced intracellular increases of [Ca(2+)](i) and chemotaxis. These results demonstrate that beta-NAD(e)(+) is an agonist of the P2Y(11) purinoceptor and that P2Y(11) is the endogenous receptor in granulocytes mediating the sustained [Ca(2+)](i) increase responsible for their functional activation.