C lotrimazole (CLT) prevents dehydration of the human HbSS red cell through inhibition of Ca++-dependent (Gardos) K+ channels in vitro (1993. J. Clin Invest. 92:520-526.) and in patients (1996. J. Clin Invest. 97:1227-1234.). Basolateral membrane K+ channels of intestinal crypt epithelial cells also participate in secretagogue-stimulated Cl- secretion. We examined the ability of CLT to block intestinal Cl- secretion by inhibition of K+ transport. Cl- secretion was measured as short-circuit current (Isc) across monolayers of T84 cells. CLT reversibly inhibited Cl- secretory responses to both cAMP- and Ca2+-dependent agonists with IC50 values of approximately 5 microM. Onset of inhibition was more rapid when CLT was applied to the basolateral cell surface. Apical Cl- channel and basolateral NaK2Cl cotransporter activities were unaffected by CLT treatment as assessed by isotopic flux measurement. In contrast, CLT strongly inhibited basolateral 86Rb efflux. These data provide evidence that CLT reversibly inhibits Cl- secretion elicited by cAMP-, cGMP-, or Ca2+-dependent agonists in T84 cells. CLT acts distal to the generation of cAMP and Ca2+ signals, and appears to inhibit basolateral K+ channels directly. CLT and related drugs may serve as novel antidiarrheal agents in humans and animals.