The role of cyclic ADP-ribose (cADPR) and its synthetic enzyme, CD38, as a downstream signal of muscarinic acetylcholine receptors (mAChRs) was examined in neuroblastoma cells expressing M1 mAChRs (NGM1). NGM1 cells were further transformed with both wild-type and mutant (C119K/C201E) human CD38. The dual transformed cells exhibited higher cADPR formation than ADPR production and elevated intracellular free Ca2+ concentrations ([Ca2+]i) in response to ACh. These phenotypes were analyzed in detail in a representative CD38 clone. The intracellular cADPR concentration by ACh application was significantly increased by CD38 overexpression. Digital image analysis by a confocal microscopy revealed that topographical distribution of the sites of Ca2+ release was unchanged between control and overexpressed cells. These results indicate that cADPR is an intracellular messenger of Ca2+ signalling, suggesting that CD38 can contribute to rnAChR-cADPR signalling. (c) 2006 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.