The role of cyclic ADP-ribose (cADPR) as the downstream signal of neuronal muscarinic acetylcholine receptors (mAChRs) and the enzyme responsible for its synthesis, ADP-ribosyl cyclase, were examined in the rat superior cervical ganglion (SCG). Application of acetylcholine or other mAChR agonists increased the ADP-ribosyl cyclase activity by about 250-300% in crude membrane fractions from the SCG of 14-day-old rats. This effect was inhibited by atropine or by the M1-mAChR antagonist, pirenzepine, and was mimicked by GTP. These results indicate that the M1 mAChRs couple to the membrane-bound form of ADP-ribosyl cyclase and suggest that cADPR is a second messenger of M1 mAChR signaling in nervous tissue. (C) 2005 Elsevier Inc. All rights reserved.