The present study was designed to determine whether the cADP-ribose-mediated Ca²⁺ signaling is involved in the inhibitory effect of nitric oxide (NO) on intracellular Ca²⁺ mobilization. With the use of fluorescent microscopic spectrometry, cADP-ribose-induced Ca²⁺ release from sarcoplasmic reticulum (SR) of bovine coronary arterial smooth muscle cells (CASMCs) was determined. In the α-toxin-permeabilized primary cultures of CASMCs, cADP-ribose (5 μM) produced a rapid Ca²⁺ release, which was completely blocked by pretreatment of cells with the cADP-ribose antagonist 8-bromo-cADP-ribose (8-Br-cADPR). In intact fura 2-loaded CASMCs, 80 mM KCl was added to depolarize the cells and increase intracellular Ca²⁺ concentration ([Ca²⁺]_i). Sodium nitroprusside (SNP), an NO donor, produced a concentration-dependent inhibition of the KCl-induced increase in [Ca²⁺]_i, but it had no effect on the U-46619-induced increase in [Ca²⁺]_i. In the presence of 8-Br-cADPR (100 μM) and ryanodine (10 μM), the inhibitory effect of SNP was markedly attenuated. HPLC analyses showed that CASMCs expressed the ADP-ribosyl cyclase activity, and SNP (1–100 μM) significantly reduced the ADP-ribosyl cyclase activity in a concentration-dependent manner. The effect of SNP was completely blocked by addition of 10 μM oxygenated hemoglobin. We conclude that ADP-ribosyl cyclase is present in CASMCs, and NO may decrease [Ca²⁺]_i by inhibition of cADP-ribose-induced Ca²⁺ mobilization.