In the present study, effects of the nitric oxide donor, S-nitroso-N-acetylpenicillamine (SNAP), on sarcoplasmic reticulum (SR) Ca2+ release were examined in freshly dissociated porcine tracheal smooth muscle (TSM) cells. Fura 2-loaded TSM cells were imaged using video fluorescence microscopy. SR Ca2+ release was induced by acetylcholine (ACh), which acts principally through inositol 1,4,5-trisphosphate (IP3) receptors, and by caffeine, which acts principally through ryanodine receptors (RyR). SNAP inhibited ACh-induced SR Ca2+ release at both 0 and 2.5 mM extracellular Ca2+. Degraded SNAP had no effect on ACh-induced SR Ca2+ release. SNAP also inhibited caffeine-induced SR Ca2+ release. ACh-induced Ca2+ influx was not affected by SNAP when SR reloading was blocked by thapsigargin. SNAP also did not affect SR Ca2+ reuptake. The membrane-permeant analogue of guanosine 3',5'-cyclic monophosphate (cGMP), 8-bromo-cGMP, mimicked the effects of SNAP. These results suggest that, in porcine TSM cells, SNAP reduces the intracellular Ca2+ response to ACh and caffeine by inhibiting SR Ca2+ release through both IP3 and RyR, but not by inhibiting influx or repletion of the SR Ca2+ stores. These effects are likely mediated via cGMP-dependent mechanisms.