Small intestinal crypt cells play a critical role in modulating Cl− secretion during digestion. The types of Cl− channels mediating Cl− secretion in the small intestine was investigated using the intestinal epithelial cell line, IEC-18, which was derived from rat small intestine crypt cells. In initial radioisotope efflux studies, exposure to forskolin, ionomycin or a decrease in extracellular osmolarity significantly increased 36Cl efflux as compared to control cells. Whole cell patch clamp techniques were subsequently used to examine in more detail the swelling-, Ca2+-, and cAMP-activated Cl− conductance. Decreasing the extracellular osmolarity from 290 to 200 mOsm activated a large outwardly rectifying Cl− current that was voltage-independent and had an anion selectivity of I− > Cl−. Increasing cytosolic Ca2+ by ionomycin activated whole cell Cl− currents, which were also outwardly rectifying but were voltage-dependent. The increase in intracellular Ca2+ levels with ionomycin was confirmed with fura-2 loaded IEC-18 cells. A third type of whole cell Cl− current was observed after increases in intracellular cAMP induced by forskolin. These cAMP-activated Cl− currents have properties consistent with cystic fibrosis transmembrane regulator (CFTR) Cl− channels, as the currents were blocked by glibenclamide or NPPB but insensitive to DIDS. In addition, the current–voltage relationship was linear and had an anion selectivity of Cl− > I−. Confocal immunofluorescence studies and Western blots with two different anti-CFTR antibodies confirmed the expression of CFTR. These results suggest that small intestinal crypt cells express multiple types of Cl− channels, which may all contribute to net Cl− secretion. © 2005 Wiley-Liss, Inc.