Nitrovasodilators relax airway smooth muscle by both guanosine 3',5'- cyclic monophosphate (cGMP)-dependent and cGMP-independent mechanisms and by mechanisms that reduce cytosolic calcium concentration ([Ca2+](i)). This study was conducted to determine the relative importance of these mechanisms in relaxation of canine tracheal smooth muscle (CTSM) induced by 3- morpholinosydnonimine (SIN-1). We measured 1) the effect of SIN-1 on force, [cGMP](i), and [Ca2+](i), and 2) the ability of methylene blue (MB) to antagonize SIN-1-induced relaxation and cGMP accumulation. The ratio of fura 2 emission fluorescence intensities due to excitation at 340- and 380-nm wavelengths (F340/F380) was used as an index of [Ca2+](i). In strips contracted with 0.3 μM acetylcholine (ACh, n = 8) or 24 mM KCl (n = 8), SIN- 1 (1-100 μM) caused a concentration-dependent decrease in force which was correlated with a concentration-dependent increase in [cGMP](i). MB (10 μM) proportionally attenuated both relaxation and cGMP accumulation. In fura 2- loaded strips contracted with 0.3 μM ACh (n = 7) or 30 mM KCl (n = 7), reductions in force induced by SIN-1 (1-100 μM) were accompanied by decreases in F340/F380. These findings suggest that in CTSM contracted with ACh or KCl, SIN-1 causes relaxation which appears to be mediated by cGMP-dependent mechanisms that reduce [Ca2+](i).