The present study determined the role of cyclic ADP-ribose (cADPR) in mediating vasoconstriction and Ca<sup>2+</sup> release in response to the activation of muscarinic receptors. Endothelium-denuded small bovine coronary arteries were microperfused under transmural pressure of 60 mm Hg. Both acetylcholine (ACh; 1 nmol/L to 1 µmol/L) and oxotremorine (OXO; 2.5–80 µmol/L) produced a concentration-dependent contraction. The vasoconstrictor responses to both ACh and OXO were significantly attenuated by nicotinamide (Nicot; an ADP-ribosyl cyclase inhibitor), 8-bromo-cADPR (8-Br-cADPR; a cADPR antagonist) or ryanodine (Ry; an Ry receptor antagonist). Intracellular Ca<sup>2+</sup> ([Ca<sup>2+</sup>]<sub>i</sub>) was determined by fluorescence spectrometry using fura-2 as a fluorescence indicator. OXO produced a rapid increase in [Ca<sup>2+</sup>]<sub>i</sub> in freshly isolated single coronary arterial smooth muscle cells (CASMCs) bathed with Ca<sup>2+</sup>-free Hanks’ solution. This OXO-induced rise in [Ca<sup>2+</sup>]<sub>i</sub> was significantly reduced by pirenzepine (PIR; an M<sub>1</sub> receptor-specific blocker), Nicot, 8-Br-cADPR or Ry. The effects of OXO on the activity of ADP-ribosyl cyclase (cADPR synthase) were examined in cultured CASMCs by measuring the rate of cyclic GDP- ribose (cGDPR) formation from β-nicotinamide guanine dinucleotide. It was found that OXO produced a concentration-dependent increase in the production of cGDPR. The stimulatory effect of OXO on ADP-ribosyl cyclase was inhibited by both PIR and Nicot. These results suggest that the cADPR signaling pathway participates in the contraction of small coronary arterial smooth muscle and Ca<sup>2+</sup> release induced by activation of M<sub>1</sub> muscarinic receptors.