Adult male rats were rendered progressively tolerant to ethanol by daily intragastric administration of doses of 3-6 g/kg body weight. Functional tolerance assessed by the hypothermic effect after injection of a challenge dose of ethanol developed slowly and was demonstrable after 2 weeks of treatment. Intrinsic crude synaptosomal membrane fluidity, as assessed by fluorescence polarization of DPH, as well as (Na+ + K+)ATPase activity, did not change significantly during the whole treatment. However, the extra addition of ethanol (0.175-0.700 M) in vitro to the membranes of rats, having received ethanol over a period of at least 2 weeks, fluidized less and inhibited the (Na+ + K+)ATPase activity less than in starch-fed controls. The time-course for the appearance of this membrane hyposensitivity was found to be the same as the time course for the development of functional tolerance to ethanol. The correlation between the degree of functional tolerance and the (Na+ + K+)ATPase sensitivity to ethanol appeared very significant, highlighting the sensitivity of membrane-bound enzymes to detecting adaptive changes in complex biological membranes tolerance.