cAMP induces both active Cl− and active K+ secretion in mammalian colon. It is generally assumed that a mechanism for K+ exit is essential to maintain cells in the hyperpolarized state, thus favoring a sustained Cl− secretion. Both Kcnn4c and Kcnma1 channels are located in colon, and this study addressed the questions of whether Kcnn4c and/or Kcnma1 channels mediate cAMP-induced K+ secretion and whether cAMP-induced K+ secretion provides the driving force for Cl− secretion. Forskolin (FSK)-enhanced short-circuit current (indicator of net electrogenic ion transport) and K+ fluxes were measured simultaneously in colonic mucosa under voltage-clamp conditions. Mucosal Na+ orthovanadate (P-type ATPase inhibitor) inhibited active K+ absorption normally present in rat distal colon. In the presence of mucosal Na+ orthovanadate, serosal FSK induced both K+ and Cl− secretion. FSK-induced K+ secretion was 1) not inhibited by either mucosal or serosal 1-[(2-chlorophenyl) diphenylmethyl]-1H-pyrazole (TRAM-34; a Kcnn4 channel blocker), 2) inhibited (92%) by mucosal iberiotoxin (Kcnma1 channel blocker), and 3) not affected by mucosal cystic fibrosis transmembrane conductance regulator inhibitor (CFTRinh-172). By contrast, FSK-induced Cl− secretion was 1) completely inhibited by serosal TRAM-34, 2) not inhibited by either mucosal or serosal iberiotoxin, and 3) completely inhibited by mucosal CFTRinh-172. These results indicate that cAMP-induced colonic K+ secretion is mediated via Kcnma1 channels located in the apical membrane and most likely contributes to stool K+ losses in secretory diarrhea. On the other hand, cAMP-induced colonic Cl− secretion requires the activity of Kcnn4b channels located in the basolateral membrane and is not dependent on the concurrent activation of apical Kcnma1 channels.