To determine whether nitric oxide (NO) formation is involved in Cl secretion across airway mucosa in response to beta adrenergic agonists, we studied the effect of isoproterenol (ISO) on the Cl diffusion potential difference of rabbit tracheal mucosa and measured NO formation by a highly specific electrode for this molecule in vivo. Perfusion of ISO on the tracheal mucosal surface increased the Cl diffusion potential difference, as determined in the presence of amiloride, in a dose-dependent fashion, the maximal increase from the base-line value being 12.1 +/- 1.7 mV (P < .001). Application of NG-nitro-L-arginine methylester (10(-3) M) decreased the Cl diffusion potential difference by itself and attenuated the subsequent response to ISO, causing a rightward displacement of ISO concentration-response curves, whereas NG-nitro-D-arginine methylester had no effect. This inhibitory effect of NG-nitro-L-arginine methyl-ester was reversed by L-arginine but not by D-arginine. Addition of ISO dose-dependently increased polarographic current and, hence, NO concentration in the perfusate, the maximal increase from the base-line levels being 178 +/- 10 nM. Histochemistry for NADPH diaphorase activity showed a strong staining within epithelial cells. These results suggest that NO formation may play a role in the beta adrenoceptor-mediated Cl secretion by tracheal mucosa.