Nitric oxide (NO) is continuously produced and released in human airways, but the biological significance of this process is unknown. In this study, we have used Calu-3 cells to investigate the effects of NO on transepithelial anion secretion. An inhibitor of NO synthase, NG-nitro-L-arginine methyl ester, reduced short- circuit current (I(sc)), whereas an NO donor, S-nitrosoglutathione (GSNO), increased I(sc), with an EC50 approximately 1.2 microM. The NO-activated current was inhibited by diphenylamine-2-carboxylate, clotrimazole, and charybdotoxin. Selective permeabilization of cell membranes indicated that NO activated both apical anion channels and basolateral potassium channels. An inhibitor of soluble guanylate cyclase, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, prevented activation of I(sc) by NO but not by 8-bromo-cGMP, suggesting that NO acts via a cGMP-dependent pathway. Sequential treatment of cells with forskolin and GSNO or 1-ethyl-2-benzimidazolinone and GSNO showed additive effects of these chemicals on I(sc). Interestingly, GSNO elevated intracellular Ca2+ concentration ([Ca2+]i) but had no effect on I(sc) activated by thapsigargin. These results show that NO activates transepithelial anion secretion via a cGMP-dependent pathway that involves cross talk between NO and [Ca2+]i.