K_VLQT1 (KCNQ1) is a voltage-gated K⁺ channel essential for repolarization of the heart action potential that is defective in cardiac arrhythmia. The channel is inhibited by the chromanol 293B, a compound that blocks cAMP-dependent electrolyte secretion in rat and human colon, therefore suggesting expression of a similar type of K⁺ channel in the colonic epithelium. We now report cloning and expression of K_VLQT1 from rat colon. Overlapping clones identified by cDNA-library screening were combined to a full length cDNA that shares high sequence homology to K_VLQT1 cloned from other species. RT-PCR analysis of rat colonic musoca demonstrated expression of K_VLQT1 in crypt cells and surface epithelium. Expression of rK_VLQT1 in Xenopus oocytes induced a typical delayed activated K⁺ current, that was further activated by increase of intracellular cAMP but not Ca²⁺ and that was blocked by the chromanol 293B. The same compound blocked a basolateral cAMP-activated K⁺ conductance in the colonic mucosal epithelium and inhibited whole cell K⁺ currents in patch-clamp experiments on isolated colonic crypts. We conclude that K_VLQT1 is forming an important component of the basolateral cAMP-activated K⁺ conductance in the colonic epithelium and plays a crucial role in diseases like secretory diarrhea and cystic fibrosis.