Cystic fibrosis (CF) is a lethal, autosomal recessive disease, caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) Cl^– channel. The most common mutation is ΔF508-CFTR, which is associated with inefficient trafficking of the mutant CFTR protein from the endoplasmic reticulum to the plasma membrane. However, because ΔF508-CFTR retains some functionality as a Cl^– channel, therapeutic efforts have been aimed at increasing the amount of ΔF508-CFTR present in the cell membrane using agents such as sodium butyrate. In this study, we examined the effects of culturing a human airway epithelial cell line, the Calu-3 cell line, in the presence of sodium butyrate (5 mM; between 0 to 96 hours). Calu-3 cells are a model of the submucosal gland serous cell, which normally express a large amount of CFTR, and have been implicated in the pathogenesis of CF pulmonary disease. When grown as monolayers, these cells exhibit basal anion secretion, further stimulated by elevation of intracellular cAMP or Ca²⁺. We report that, over the time period investigated, butyrate treatment causes a significant decrease in Ca²⁺-activated Cl^– secretion, measured as the change in transepithelial short circuit current in response to the Ca²⁺ elevating agent thapsigargin (control = 153.8 ± 2.1 μA cm^-2 (mean ± S.E.M.) (n = 3); 96 h butyrate = 7.9 ± 4.3 μA cm^-2 (n = 3); P < 0.0001, two-tailed t test). Additionally, there is a corresponding loss of response to the secretagogue 1-EBIO, an agent which activates the intermediate conductance, Ca²⁺-activated basolateral K⁺ channel encoded by KCNN4. Initial baseline short circuit current also decreased over the same period of time (from control value of 35.6 ± 3.4 μA cm^-2 (n =3) to 6.6 ± 0.03 μA cm^-2 (n = 4) following 96 h butyrate treatment; P < 0.001). Quantitative PCR revealed that these losses of response are associated with dramatic decreases in the level of mRNA for both KCNN4 and CFTR. These results suggest that prolonged exposure to sodium butyrate results in a significant down-regulation of KCNN4 and CFTR gene expression, and most importantly, a functional loss of Ca²⁺-stimulated transepithelial anion secretion. However, since only wild type CFTR was studied, we cannot preclude the possibility that butyrate may have different effects on the expression of ΔF508-CFTR. Furthermore, we suggest that the major component of basal transepithelial anion secretion in unstimulated Calu-3 cells is supported by basal KCNN4 channel activity. Therefore, long term exposure to sodium butyrate may ultimately exacerbate rather than ameliorate the anion secretory deficit seen in CF, especially in tissues that normally exhibit Ca²⁺-activated anion secretion.