The alteration of resting tension (RT) from 0.5 g to 2.5 g increased significantly airway smooth muscle contractions induced by acetylcholine (ACh) in rabbit trachea. The decrease in extracellular calcium concentration [Ca2+]o from 2 mM to 0.2 mM reduced ACh-induced contractions only at 2.5 g RT with no effect at 0.5 g RT. The nonselective inhibitor of nitric oxide synthase (NOS), N(G)-nitro-L-arginine methyl ester (L-NAME) increased ACh-induced contractions at 2.5 g RT. The inhibitor of inducible NOS, S-methylsothiourea or neuronal NOS, 7-nitroindazole had no effect. At 2.5 g RT, the reduction of [Ca2+]o from 2 mM to 0.2 mM abolished the effect of L-NAME on ACh-induced contractions. The NO precursor L-arginine or the tyrosine kinase inhibitors erbstatin A and genistein had no effect on ACh-induced contractions obtained at 2.5 g RT. Our results suggest that in airways, RT affects ACh-induced contractions by modulating the activity of epithelial NOS in a calcium-dependent, tyrosine-phosphorylation-independent way.