The effects of chronic ethanol consumption on the temperature dependence of the motion, order, and partition of spin probes were investigated in both intact rat mitochondrial membranes and phospholipid vesicles derived from those membranes. In intact membranes from chronic alcoholic animals an abrupt change in the order and motion parameters (5 doxylstearic acid), as well as in the partition coefficient of 5-doxyldecane (5N10), occurs at about 26 °C, compared to 20 °C in the control. This shift in temperature parallels the shifts in Arrhenius plots of respiration and ATPase activities previously reported ( H. Rottenberg, D. E. Robertson, and E. Rubin, 1980, Lab. Invest. 42, 318–326 ). Similar shifts are observed in vesicles prepared from extracted phospholipids, thus confirming the suggestion that these changes result from an alteration in molecular ordering of the lipid structure. Membranes and phospholipid vesicles from chronic alcoholic rats show significantly higher order parameters. Similarly, the partition coefficient of the nitroxydecane, 5N10, is lower in membranes from chronic alcoholic animals over the entire temperature range. These findings indicate increased rigidity of these membranes. While ethanol, in vitro, greatly enhances the partition of 5N10 into normal membranes, it has almost no effect on its partition into the membranes from chronic alcoholic rats. These findings, together with our recent observation that, at high temperature, membranes from chronic alcoholic animals are resistant to the disordering effect of ethanol ( A. J. Waring, H. Rottenberg, T. Ohnishi, and E. Rubin, 1981, Proc. Nat. Acad. Sci. USA 78, 2582–2586 ), may explain the resistance of these membranes to uncoupling by ethanol. We suggest that an adaptive adjustment of the phospholipid composition of mitochondrial membranes causes increased rigidity, which is manifested by shifts in the temperature dependence of the physical properties of the mitochondrial membranes from ethanol-fed rats. Although these adjustments tend to preserve adequate coupling efficiency in chronic alcoholic rats, they may also lead to many of the ethanol-induced mitochondrial alterations previously reported.