We have investigated the interaction of clotrimazole (CLT) and related compounds with the erythroid Ca(2+)-activated K+ channel, a mediator of sickle cell dehydration. We measured K+ transport, membrane potential, and cell volume upon activation of this pathway in sickle erythrocytes. CLT blocked almost completely Ca(2+)-activated K+ transport in homozygous hemoglobin S cells, with IC50 values of 29 +/- 15 nM in isotonic 20 mM salt solution and 51 +/- 15 nM in normal saline (n = 3). The inhibition of K+ transport by CLT was caused by a specific interaction with the Ca(2+)-activated K+ channel of human red cells, since it displaced bound 125I-Charybdotoxin, a specific ligand of the Gardos channel, with an IC50 (12 +/- 4 nM in isotonic 20 mM) similar to the IC50 values for flux inhibition. When homozygous hemoglobin S cells were dehydrated by incubation in the presence of 100 microM CaCl2 and the ionophore A23187, or by exposure to cycles of oxygenation and deoxygenation, CLT effectively inhibited cell dehydration and K+ loss. The IC50 of CLT for inhibition of Ca(2+)-activated K+ transport in sickle cells is significantly lower than plasma concentrations of CLT achievable after nontoxic oral doses. We therefore propose that oral administration of CLT may prevent red cell dehydration in patients with sickle cell anemia.