Phenotypic acclimation is generally assumed to confer an advantage in the environment that stimulates the response. To test this beneficial acclimation assumption explicitly, we investigated the consequences of temperature acclimation for the fitness of Escherichia coli at two temperatures, 32 degrees C and 41.5 degrees C. Both temperatures permit growth and long-term persistence of the genotypes in serial culture. We found that prior acclimation to 32 degrees C, relative to acclimation to 41.5 degrees C, enhanced fitness at 32 degrees C, consistent with the assumption. But prior acclimation to 41.5 degrees C actually reduced fitness at 41.5 degrees C, relative to acclimation to 32 degrees C. Hence, the assumption that acclimation always confers an advantage is demonstrated to be false. Acclimation to 41.5 degrees C did, however, improve survival at 50 degrees C, a lethal temperature. This protective response has been shown to be associated with the induction of stress proteins. The reduced competitive fitness caused by acclimation at 41.5 degrees C may reflect a physiological burden associated with expression of stress proteins when they are not needed to prevent lethal damage. Whatever the cause, acclimation to the higher temperature decreased competitive fitness at that temperature.