To measure the generation of nitric oxide (NO) by airway epithelial cells and to study the physiological role of NO in the regulation of epithelial functions, we studied ciliary motility of rabbit cultured tracheal epithelium in vitro and ion transport across tracheal mucosa in vivo. Isoproterenol dose-dependently increased ciliary beat frequency, as measured by photoelectric methods. Perfusion of tracheal mucosa with tachykinins increased the diffusion potential for chloride ions, as measured in the presence of amiloride under open-circuit conditions: the rank order of potency was neurokinin A > substance P >> neurokinin B. These responses to isoproterenol and to tachykinins were attenuated by pretreatment with NG-nitro-L-arginine methylester, and this attenuation was reversed by L-arginine. In contrast, NG-nitro-D-arginine methylester and D-arginine had no such effects. NO concentrations in the medium and perfusate were measured in realtime with an amperometric sensor specific to this molecule, and immersion of the electrode allowed detections of a polarographic current under unstimulated conditions. Addition of isoproterenol, neurokinin A, or substance P caused a rapid and dose-dependent increase in NO concentration. Histochemical examination for NADPH diaphorase activity in cultured epithelium showed strong staining within the cytoplasm. These results suggest that NO is spontaneously released from airway epithelial cells and that generation of this molecule may contribute to airway epithelial ciliary motility and chloride ion secretion mediated by beta-adrenoceptors and by tachyinin NK2 receptors.