Stimulation of muscarinic receptors in the duodenal mucosa raises cytosolic free Ca²⁺ ([Ca²⁺]_cyt), thereby regulating duodenal epithelial ion transport. However, little is known about the downstream molecular targets that account for this Ca²⁺-mediated biological action. Ca²⁺-activated K⁺ channels (K_Ca) are candidates, but the expression and function of duodenal K_Ca are poorly understood. Therefore, we determined whether K_Ca are expressed in the duodenal mucosa and their involvement in Ca²⁺-mediated duodenal epithelial ion transport. Two selective blockers of intermediate Ca²⁺-activated K⁺ channels (IK_Ca), clotrimazole (30 µM) and 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34, 10 µM), significantly inhibited carbachol (CCh)-induced both duodenal short-circuit current (I_sc) and duodenal mucosal bicarbonate secretion (DMBS) in mice, but did not affect responses to forskolin and heat-stable enterotoxin of Escherichia coli. Tetraethylammonium, 4-aminopyridine, and BaCl₂ failed to inhibit CCh-induced I_sc and DMBS. A23187 (10 µM), a Ca²⁺ ionophore, and 1-ethyl-2-benzimidazolone (1-EBIO, 1 mM), a selective opener of K_Ca, increased both I_sc and DMBS. The effect of 1-EBIO was more pronounced in serosal than mucosal addition. Again, both clotrimazole and TRAM-34 significantly reduced A23187- or 1-EBIO-induced I_sc and DMBS. Moreover, clotrimazole (20 mg/kg, i.p.) significantly attenuated acid-stimulated DMBS of mice in vivo. Finally, the molecular identity of IK_Ca was verified as KCNN4 (SK4) in freshly isolated murine duodenal mucosae by RT-PCR and Western blotting. Taken together, our results suggest that IK_Ca is one of the downstream molecular targets for [Ca²⁺]_cyt to mediate duodenal epithelial ion transport.