The relative stability against a decrease in adrenergic stimulation of the uncoupling protein (UCP) incorporated into different mitochondrial fractions was investigated in brown-fat-cell cultures. Cultures were initiated with undifferentiated cells from young mice and were acutely stimulated with noradrenaline at confluence (day 7). Cells were harvested just after the finish of the 24 h stimulation treatment or 24 h later, and three mitochondrial fractions were isolated by differential centrifugation: the M1 fraction (1000 g), the M3 fraction (3000 g) and the M15 fraction (15,000 g). The results obtained in vitro indicate that removal of adrenergic stimulation determines a selective loss of UCP from the lightest mitochondrial fractions (M3 and M15). Similar results were obtained in a situation in vivo (24 h starvation in mice) which is known to lead to a decreased noradrenaline input to brown adipose tissue, with decreased UCP levels. Thus brown adipocytes possess different mitochondrial subpopulations, which exhibit characteristic changes in their UCP turnover in response to thermogenic signals.