In this study we investigated the dependence of Na⁺ transport regulation on membrane cholesterol content in A6 epithelia. We continuously monitored the short-circuit current (I_sc), the transepithelial conductance (G_T) and the transepithelial capacitance (C_T) to evaluate the effects of cholesterol extraction from the apical and basolateral membranes in steady-state conditions and during activation with a hypo-osmotic shock, oxytocin and adenosine. Cholesterol extraction was achieved by perfusing the epithelia with methyl--cyclodextrin (mCD) for 1h. In steady-state conditions, apical membrane cholesterol extraction did not significantly affect the electrophysiological parameters, in contrast to the marked reductions observed during basolateral mCD treatment. However, apical mCD application hampered the responses of I_sc and G_T to hypotonicity, oxytocin and adenosine. Analysis of the blocker-induced fluctuation in current demonstrated that apical mCD treatment decreased the epithelial sodium channel open probability in steady-state as well as after activation of Na⁺ transport by adenosine, whereas the density of conducting channels was not significantly changed, as confirmed by C_T measurements. Na⁺ transport activation by hypotonicity was abolished during basolateral mCD treatment due to a reduced Na⁺/K⁺ pump activity. From this study we conclude that basolateral membrane cholesterol extraction reduces Na⁺/K⁺ pump activity, whereas the reduced cholesterol content of the apical membranes affects the activation of Na⁺ transport by reducing the sodium channel open probability.