The purpose of the current experiments was 1) to assess basolateral Na⁺-K⁺-2Cl cotransporter (NKCC1) expression and 2) to ascertain the role of cystic fibrosis transmembrane conductance regulator (CFTR) in the regulation of this transporter in a prototypical pancreatic duct epithelial cell line. Previously validated human pancreatic duct cell lines (CFPAC-1), which exhibit physiological features prototypical of cystic fibrosis, and normal pancreatic duct epithelia (stable recombinant CFTR-bearing CFPAC-1 cells, termed CFPAC-WT) were grown to confluence before molecular and functional studies. High-stringency Northern blot hybridization, utilizing specific cDNA probes, confirmed that NKCC1 was expressed in both cell lines and its mRNA levels were twofold higher in CFPAC-WT cells than in CFPAC-1 cells (P < 0.01, n = 3). Na⁺-K⁺-2Cl cotransporter activity, assayed as the bumetanide-sensitive, Na⁺- and Cl-dependent NH⁺₄ entry into the cell (with NH⁺₄ acting as a substitute for K⁺), increased by ~115% in CFPAC-WT cells compared with CFPAC-1 cells (P < 0.01, n = 6). Reducing the intracellular Cl by incubating the cells in a Cl-free medium increased Na⁺-K⁺-2Cl cotransporter activity by twofold (P < 0.01, n = 4) only in CFPAC-WT cells. We concluded that NKCC1 is expressed in pancreatic duct cells and mediates the entry of Cl. NKCC1 activity is enhanced in the presence of an inward Cl gradient. The results further indicate that the presence of functional CFTR enhances the expression of NKCC1. We speculate that CFTR regulates this process in a Cl-dependent manner.