cholesterol depletion alters the apparent affinity of the internal cationic sites and the maximal translocation rate but not the affinity of the external cationic sites of the Na+-K+ pump in human erythrocytes. To test whether these effects were mediated by a direct cholesterol-internal site interaction or by a change in membrane lipid order, the effects of five fluidizing amphiphiles (chlorpromazine, imipramine, benzyl alcohol, sodium oleate and sodium benzenesulphonate) on the kinetic parameters of the Na+-K+ pump were determined. The cholesterol removal and all the agents used induced dose-response decreases in membrane lipid order as measured by fluorescence polarization or ESR. Positive and neutral amphiphiles mimicked the effects of cholesterol removal on the affinity of the internal sites of the pump and to a lesser extent on the maximal translocation rate. Anionic amphiphiles had no effect on internal sites, probably because they distributed preferentially within the outer leaflet on the membrane. These results indicate that cholesterol controls the affinity of the internal sites of the Na+-K+ pump by altering the membrane lipid order. In contrast, neither cholesterol depletion nor the agents used altered the affinity of the external sites of the Na+-K+ pump. This difference in sensitivity to membrane lipids order suggests that internal and external cationic sites, although borne by the same protein, are in different lipid environments.