Nitric oxide is produced during intestinal inflammation and inhibits the epithelial responsiveness to cAMP-dependent secretagogues. The effect is presumably due to inhibition of activation of the cystic fibrosis transmembrane conductance regulator (CFTR). However, since insertion of CFTR into the epithelial apical membrane is also a cAMP-dependent process, we tested the hypothesis that NO could inhibit cAMP-dependent CFTR trafficking. SCBN intestinal epithelial cells were treated with forskolin to activate adenylate cyclase activity. The cells were fixed at various times and immunostained for CFTR. Some cells were pretreated with the nitric oxide donor, PAPA-NONOate, the protein kinase A inhibitor, H89, or the microtubule blocker, nocodazole. Cross sections of epithelial monolayers were then studied under fluorescence and the ratio of apical to basolateral CFTR immunoreactivity determined. Stimulation of adenylate cyclase activity caused an increase in the apical:basolateral ratio of CFTR within 30 seconds. This effect was transient and preceded changes in short circuit current in SCBN monolayers mounted in Ussing chambers. PAPA-NONOate, H89 and nocodazole all reduced forskolin-stimulated CFTR trafficking. The inhibitory effect of the NO donor was not affected by pretreatment with the guanylate cyclase inhibitor, ODQ. PAPA-NONOate reduced forskolin-stimulated increases in intracellular cAMP. The data suggest that a portion of the inhibitory effect of nitric oxide donors on cAMP-dependent chloride secretion is through the inhibition of cAMP-dependent insertion of CFTR into the apical plasma membrane. These data provide insight into the mechanism of secretory dysfunction in inflammatory diseases of the gut where mucosal nitric oxide is elevated.