Epithelial Cl- channels are regulated by various physiological factors, including guanosine 3',5'-cyclic monophosphate (cGMP). Because cGMP mediates many of the physiological actions of nitric oxide (NO), we have studied both the presence of endogenous NO and the effects of exogenous NO on Cl- currents in A549 human lung epithelial cells. We have detected Ca(2+)-dependent NO synthase activity in A549 cells. Using the perforated patch-clamp technique, we have shown that inhibition of this enzyme by NG-monomethyl-L-arginine decreased Cl- current, an effect that was reversed by the NO donor S-nitrosoglutathione (GSNO). In addition, the NO donors GSNO and S-nitroso-N-acetyl-D,L-penicillamine increased whole-cell Cl- currents in A549 cells. This stimulatory effect of the NO donors was sensitive to inhibition by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, suggesting that channels other than the cystic fibrosis transmembrane conductance regulator (CFTR) are involved in the action of NO on A549 cells. In addition, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, a selective inhibitor of soluble guanylyl cyclase, decreased NO-mediated stimulation of Cl- currents. Our results suggest that, in lung epithelial cells, NO regulates a non-CFTR Cl- conductance acting via a cGMP-dependent mechanism.