Human red blood cells (R.B.C.) were incubated with phosphatidylcholine vesicles to obtain partial depletion of their membrane cholesterol. The kinetic parameters of the Na pump (affinities and maximal fluxes) were determined in these cells and compared with controls. 2. In the presence of internal K, progressive cholesterol depletion induces gradually (i) an increase of the maximal flux (ii) a reduction of the apparent affinity for internal Na. 3. In the absence of internal K, cholesterol depletion has no apparent effect, indicating that the observed changes were mediated by the internal K. Cholesterol depletion acts by increasing these two K effects: (i) activation of the maximal fluxes, and (ii) reduction of the internal Na affinity. 4. The cholesterol effect (on the K-pump interaction) is specific; it occurs without any change (i) in the affinities for internal Na and inorganic phosphate and for external Na and K, and (ii) in the number of pumping sites. 5. Under physiological conditions, cholesterol reduces the cation translocation rate and increases the selectivity of the pump for internal Na.
