The antifungal antibiotic clotrimazole (CLT) blocks directly and with high potency the Ca2+-activated K+ channels of human erythrocytes, erythroleukemia cells, and ferret vascular smooth muscle cells. We recently reported that CLT inhibits Cl- secretion in human intestinal T84 cells, likely by affecting K+ transport (Rufo, P.A., L. Jiang, S.J. Moe, C. Brugnara, S.L. Alper, and W.I. Lencer. 1996. J. Clin. Invest. 98:2066-2075). To determine if CLT had direct effects on K+ conductances in T84 cells, we selectively permeabilized apical membranes of confluent T84 cell monolayers using the ionophore amphotericin B. This technique permits direct measurement of basolateral K+ transport. We found that CLT and a stable des-imidazolyl derivative inhibited directly two pharmacologically distinct basolateral membrane K+conductances, but had no effect on apical membrane Cl- conductances. The effects of CLT on Cl- secretion were also examined in intact tissue. CLT inhibited forskolin-induced Cl- secretion in rabbit colonic mucosal sheets mounted in Ussing chambers by 91%. CLT also inhibited cholera toxin-induced intestinal Cl- secretion in intact mice by 94%. These data provide direct evidence that CLT blocks Cl- secretion in intestinal T84 cells by inhibition of basolateral K+ conductances, and show that CLT inhibits salt and water secretion from intact tissue in vitro and in vivo. The results further support the suggestion that CLT and its metabolites may show clinical efficacy in the treatment of secretory diarrheas of diverse etiologies.