We investigated the role of the membrane cholesterol content on the regulation of Na⁺ transport activation in A6 renal epithelia. Voltage-clamped epithelia were continuously monitored for changes in short-circuit current (I_sc), transepithelial conductance (G_T) and capacitance (C_T). Cholesterol was extracted from either the apical or the basolateral membranes by local perfusion with methyl-b-cyclodextrin (mbCD, 10 mM) for 1 h. Subsequently, Na⁺ transport was activated by applying adenosine (1 mM) to the basolateral border. Apical membrane cholesterol extraction did not affect the basal levels of Na⁺ transport, but attenuated the increase in amiloride-sensitive I_sc elicited by basolateral adenosine with 47%. The reduced levels of cholesterol in the apical membrane did not affect the 10% rise in C_T, pointing to an intact process of channel insertion. Analysis of the fluctuation in current induced by 6-chloro-3,5-diaminopyrazine-2-carboxamide (CDPC) revealed a decrease in Na⁺ channel open probability (ENaC P_o) at the apical membrane, whereas the number of Na⁺ conducting channels approximately doubled in both conditions. Basolateral membrane cholesterol extraction already reduced the basal levels of Na⁺ transport. In addition, the reduced basolateral membrane cholesterol content completely abolished the activation of Na⁺ transport in response to a hyposmotic shock. Impedance analysis revealed that basolateral membrane cholesterol extraction reduced the activity of the Na⁺/K⁺-ATPase. Interestingly, cholesterol extraction from the basolateral membrane did not affect the regulatory volume decrease, indicating that transporters involved in volume regulation were still intact in these conditions.