Cell dehydration mediated by Ca²⁺-activated K⁺ channels plays an important role in the pathogenesis of sickle cell disease. CD-1 mouse erythrocytes possess a Ca²⁺-activated K⁺ channel (Gardos channel) with maximal velocity (V _max) of 0.154 ± 0.02 mmol ⋅ l cells⁻¹ ⋅ min⁻¹and an affinity constant (K _0.5) for Ca²⁺ of 286 ± 83 nM in the presence of A-23187. Cells pretreated with 500 nM endothelin-1 (ET-1) increased theirV _max by 88 ± 9% (n = 8) and decreased theirK _0.5 for Ca²⁺ to 139 ± 63 nM (P < 0.05;n = 4). Activation of the Gardos channel resulted in an EC₅₀ of 75 ± 20 nM for ET-1 and 374 ± 97 nM for ET-3. Analysis of the affinity of unlabeled ET-1 for its receptor showed two classes of binding sites with apparent dissociation constants of 167 ± 51 and 785 ± 143 nM and with capacity of binding sites of 298 ± 38 and 1,568 ± 211 sites/cell, respectively. The Gardos channel was activated by the endothelin B (ET_B) receptor agonist IRL 1620 and inhibited by BQ-788, demonstrating the involvement of ET_B receptors. Calphostin C inhibited 73% of ET-1-induced Gardos activation and 84% of the ET-1-induced membrane protein kinase C activity. Thus endothelins regulate erythrocyte Gardos channels via ET_B receptors and a calphostin-sensitive mechanism.