To determine whether nitric oxide (NO) regulates mucus secretion from airway submucosal glands which are the main source of human airway secretion, we examined the effects of NO synthase inhibitors (L-NAME and L-NMMA) on mucus glycoprotein (MGP) secretion from feline and human airway explants (with epithelium) and isolated submucosal glands. MGP secretion was estimated by measuring trichloroacetic-acid (TCA) precipitable [H3]-glycoconjugates using secretory indices. NO synthase inhibitors alone did not alter significantly MGP secretion from explants or isolated glands. Pretreatment with NO synthase inhibitors significantly inhibited both methacholine (MCh) and bradykinin (BK)-induced secretion from isolated glands, but not significantly inhibit the secretion from explants. The inhibition by L-NAME was reversed by the addition of L-arginine in both MCh- and BK-induced secretions from isolated glands. Further, a NO generator isosorbide dinitrate induced a significant increase in the secretion. These findings suggest that endogenous NO has a stimulatory action in airway submucosal gland secretion and directly regulates the secretion from submucosal glands independently of superficial epithelial cells.