Ca²⁺-dependent anion secretion in distal colon requires the simultaneous activity of apical CFTR Cl^– channels activated by cAMP and basolateral (BL) K⁺ channels activated by Ca²⁺. Three classes of Ca²⁺-activated K⁺ channels have been distinguished from their single channel conductances. The small-, intermediate-, and large-conductance channels SK, IK, and BK, respectively. SK channels are encoded by three genes, Kcnn1-3, IK and BK channels are the products of the Kcnn4 and Slo (Kcnma1) genes, respectively. Ca²⁺-activated K⁺ channels of all three types have been identified in the colon and could participate in Ca²⁺-dependent anion secretion. We have now used an animal deficient in IK1, the Kcnn4 null mouse (Begenesich et al., 2004), to test for its participation in this process. A piece of distal colon was gently stripped of the muscle layer, placed in an Ussing chamber (0.1 cm2) in a bicarbonate buffered solution, gassed with 95% O2/5% CO2 at 37°C. The transepitheial potential difference was monitored continuously and 20 μA pulses were passed to calculate tissue resistance and equivalent short circuit current (Isc). To induce cAMP-mediated Cl^– secretion tissues were incubated with 100 μM IBMX and 1 μM forskolin. Chromanol 293 B (10 μM) was added at the BL side to close KCNQ1/KCNE3 K+ channels. BL carbachol (CCh, 100 μM) was used as Ca²⁺ agonist. Clotrimazol (3 μM) and Ba²⁺ (5 mM) were used as K⁺ channel blockers. For K⁺ secretion experiments, tissues were incubated with indomethacin (2 μM) previous to CCh application, to avoid basal release of prostaglandin and consequent cAMP increase. Wild type (WT) and knockout (KO) animals had cAMP-induced Cl^– secretion of similar magnitude (ΔIsc -149 ± 21 and -144 ± 11 μA cm-2, means ± SEM, n=5 & 4 respectively). When CCh was added, only WT tissues had a current response consistent with Ca²⁺ dependent Cl^– secretion (ΔIsc = -174 ± 12 μA cm-2), which was decreased by the IK1 inhibitor clotrimazol (ΔIsc = -38 ± 3.6 μA cm^-2, n=5, p<0.0005). In KO tissues, CCh induced positive Isc change (ΔIsc 17 ± 6 μA cm^-2, n=4) consistent with K⁺ secretion, which was inhibited by apical Ba²⁺ (ΔIsc 8.7 ± 1.8 -2, n=3, p<0.05), but not by clotrimazol (ΔIsc 19 ± 7 μA cm^-2, n=3). Experiments with epithelia treated with indomethacin suggest that both WT and KO tissues are able to secrete K⁺ under CCh stimulation. This was inhibited by Ba²⁺ but not affected by clotrimazol. Our data suggests that IK1 encoded by the Kcnn4 gene is the main Ca²⁺-activated K⁺ channel that drives Cl^– secretion by Ca²⁺ agonists in mouse distal colon. They also suggest that a different, as yet unidentified Ba²⁺-sensitive apical K⁺ channel mediates CCh-activated K⁺ secretion.