Nitric oxide (·NO) has been implicated in a wide range of autocrine and paracrine signaling mechanisms. Herein, we assessed the role of exogenous ·NO in the modulation of heterologous gene expression in polarized kidney epithelial cells (LLC-PK1) that were stably transduced with a cDNA encoding human wild-type cystic fibrosis transmembrane conductance regulator (CFTR) under the control of a heavy metal-sensitive metallothionein promoter (LLC- PK1-WTCFTR). Exposure of these cells to 125 μM DETA NONOate at 37°C for 24 h (a chemical ·NO donor) diminished Zn2+-induced and uninduced CFTR protein levels by 43.3 ± 5.1 and 34.4 ± 17.1% from their corresponding control values, respectively. These changes did not occur if red blood cells, effective scavengers of ·NO, were added to the medium. Exposure to ·NO did not alter lactate dehydrogenase release in the medium or the extent of apoptosis. Coculturing LLC-PK1-WTCFTR cells with murine fibroblasts that were stably transduced with the human inducible ·NO synthase cDNA gene also inhibited CFTR protein expression in a manner that was antagonized by 1 mM N(G)-monomethyl-L-arginine in the medium. Pretreatment of LLC-PK1-WTCFTR with ODQ, an inhibitor of guanylyl cyclase, did not affect the ability of ·NO to inhibit heterologous CFTR expression; furthermore, 8-bromo-cGMP had no effect on heterologous CFTR expression. These data indicate that ·NO impairs the heterologous expression of CFTR in epithelial cells at the protein level via cGMP-independent mechanisms.