Secretion of Cl− and K+ in the colonic epithelium operates through a cellular mechanism requiring K+ channels in the basolateral and apical membranes. Transepithelial current [short-circuit current (Isc)] and conductance (Gt) were measured for isolated distal colonic mucosa during secretory activation by epinephrine (Epi) or PGE2 and synergistically by PGE2 and carbachol (PGE2 + CCh). TRAM-34 at 0.5 μM, an inhibitor of KCa3.1 (IK, Kcnn4) K+ channels (H. Wulff, M. J. Miller, W. Hänsel, S. Grissmer, M. D. Cahalan, and K. G. Chandy. Proc Natl Acad Sci USA 97: 8151–8156, 2000), did not alter secretory Isc or Gt in guinea pig or rat colon. The presence of KCa3.1 in the mucosa was confirmed by immunoblot and immunofluorescence detection. At 100 μM, TRAM-34 inhibited Isc and Gtactivated by Epi (∼4%), PGE2 (∼30%) and PGE2 + CCh (∼60%). The IC50 of 4.0 μM implicated involvement of K+ channels other than KCa3.1. The secretory responses augmented by the K+ channel opener 1-EBIO were inhibited only at a high concentration of TRAM-34, suggesting further that KCa3.1 was not involved. Sensitivity of the synergistic response (PGE2 + CCh) to a high concentration TRAM-34 supported a requirement for multiple K+ conductive pathways in secretion. Clofilium (100 μM), a quaternary ammonium, inhibited Cl− secretory Iscand Gt activated by PGE2 (∼20%) but not K+ secretion activated by Epi. Thus Cl−secretion activated by physiological secretagogues occurred without apparent activity of KCa3.1 channels but was dependent on other types of K+ channels sensitive to high concentrations of TRAM-34 and/or clofilium.