Many hormones and neurotransmitters evoke Ca²⁺ release fromintracellular stores, often triggering agonist-specific signatures of intracellular Ca²⁺ concentration^,,,,. Inositol trisphosphate (InsP₃)¹ and cyclic adenosine 5′-diphosphate-ribose (cADPR), are established Ca²⁺-mobilizing messengers that activate Ca²⁺ release through intracellular InsP₃ and ryanodine receptors, respectively^,,. However, in pancreatic acinar cells, neither messenger can explain the complex pattern of Ca²⁺ signals triggered by the secretory hormone cholecystokinin (CCK). We show here that the Ca²⁺-mobilizing molecule nicotinic acid adenine dinucleotide phosphate (NAADP),^,,,,, an endogenous metabolite of β-NADP, triggers a Ca²⁺ response that varies from short-lasting Ca²⁺ spikes to a complex mixture of short-lasting (1–2 s) and long-lasting (0.2–1 min) Ca²⁺ spikes. Cells were significantly more sensitive to NAADP than to either cADPR or InsP₃, whereas higher concentrations of NAADP selectively inactivated CCK-evoked Ca²⁺ signals in pancreatic acinar cells, indicating that NAADP may function as an intracellular messenger in mammalian cells.