The temperature dependence of lactose active transport, efflux down a concentration gradient, and equilibrium exchange were analyzed in right-side-out membrane vesicles from Escherichia coli containing wild-type lactose permease and mutant Glu325 f Ala. With respect to uphill transport and efflux down a concentration gradient, both of which involve H+ symport, Arrhenius plots with wild-ype permease exhibit a discontinuity at 18-19℃ with a 7-8-fold decrease in activation energy above the phase transition. For equilibrium exchange, which does not involve H+ symport, the change in activation energy is much less pronounced (2-3-fold) than that observed for active transport or efflux. Strikingly, mutant Glu325f Ala, which catalyzes equilibrium exchange as well as wild-type permease but is defective in all translocation reactions that involve net H+ translocation, exhibits no change whatsoever in activation energy. The findings are consistent with the conclusion that the primary effect of the lipid phase transition is to alter coupling between substrate and H+ translocation rather than the conformational change (s) responsible for translocation across the membrane.