We have reported previously that a cold acclimation procedure (3-hr partial restraint at 6 degrees C, repeated 3 times at 2-week intervals) usually improves the cold tolerance of adult C57BL/6J mice. Those mice that did not improve their cold tolerance had lower cold-induced sympathetic nervous outflow to the interscapular brown adipose tissue (IBAT), suggesting a failure in the mechanisms of nonshivering thermogenesis. To understand the origin of this failure, this study was intended to measure nonshivering thermogenesis in mice that did not improve their cold tolerance after the cold acclimation procedure. After being subjected 3 times to a partial restraint at 6 degrees C, mice were anesthetized with urethane, immobilized with vecuronium bromide, and placed on artificial ventilation. The VO2 and VCO2 in expired air were measured and metabolic heat production (MHP) was calculated while body temperature was artificially lowered to 7.5 degrees C below control level. In a separate group of mice, the total amount and concentration of mitochondrial uncoupling protein, thermogenin (UCP), in IBAT was measured immediately after completion of the cold-acclimation procedure. The concentration and the amount of UCP in the mitochondria of IBAT was significantly higher in all mice that had been presented to the cold acclimation procedure, regardless of its outcome, than in mice that had never been exposed to an environment below room temperature (NAIVE). MHP increased significantly during body cooling in all mice. However, MHP before and during cold stimulation in mice that did not improve their cold tolerance as a result of the cold-acclimation procedure was significantly lower than the MHP of animals in which cold tolerance was improved, and was not different from MHP of the NAIVE group. Therefore, in mice in which cold tolerance did not improve after repeated cold exposure, the anatomical and biochemical changes in brown adipose tissue typical of cold acclimation were not associated with a cold-induced increase in MHP. We infer that the expression of UCP in brown adipose tissue is a necessary, but not sufficient, attribute of cold acclimation. Cold acclimation, measured as increased cold tolerance, occurs only if synthesis of UCP in BAT is associated with an increased cold-induced response of the sympathetic nervous system.