We showed that muscarinic acetylcholine (ACh)-stimulation increased the cellular content of cADPR in the pancreatic acinar cells from normal mice but not in those from CD38 knockout mice. By monitoring ACh-evoked increases in the cytosolic Ca2+ concentration ([Ca2+]i) using fura-2 microfluorimetry, we distinguished and characterized the Ca2+ release mechanisms responsive to cADPR. The Ca2+ response from the cells of the knockout mice (KO cells) lacked two components of the muscarinic Ca2+ release present in wild mice. The first component inducible by the low concentration of ACh contributed to regenerative Ca2+ spikes. This component was abolished by ryanodine treatment in the normal cells and was severely impaired in KO cells, indicating that the low ACh-induced regenerative spike responses were caused by cADPR-dependent Ca2+ release from a pool regulated by a class of ryanodine receptors. The second component inducible by the high concentration of ACh was involved in the phasic Ca2+ response, and it was not abolished by ryanodine treatment. Overall, we conclude that muscarinic Ca2+ signaling in pancreatic acinar cells involves a CD38-dependent pathway responsible for two cADPR-dependent Ca2+ release mechanisms in which the one sensitive to ryanodine plays a crucial role for the generation of repetitive Ca2+ spikes.