The role of calmodulin in stimulating active calcium transport in the human red cell membrane is well documented. In contrast, efforts to characterize the effect of calmodulin on the Ca2+-dependent K+ channel in erythrocyte membranes have given rise to conflicting reports. These studies have indicated that experimental conditions may play a critical role in preserving the Ca2+-dependent K+ channels in erythrocyte inside-out vesicles. With these observations in mind, a double-labelling study of simultaneous active Ca2+ and passive Rb+ uptake in red-cell inside-out vesicles was undertaken. Addition of calmodulin and ATP to a suspension of inside-out vesicles containing 1 mM K+ caused a Ca2+-dependent increase in both the rate of active calcium transport and Rb+ uptake. The initial Rb+ isotope flux was increased 3-fold over the rate observed in the absence of calmodulin. The k1/2 for activation of K+ permeability was approx. 5 X 10(-7) M Ca2+ as compared to 10(-6) M Ca2+ for active Ca2+ transport. Addition of the calmodulin antagonists pimozide and chlorpromazine blocked calmodulin activation of the Ca2+-dependent K+ channel. The observation that activation of the K+ channel occurs at Ca2+ concentrations which are lower than those required for maximum stimulation of the calcium pump suggests that these processes are dependent on two states of the calmodulin molecule, characterized by a lower or higher amount of Ca2+ bound to calmodulin.