The effects of starvation on the thermogenic parameters of three different mitochondrial subpopulations in brown adipose tissue (BAT) of both post-cafeteria obese and lean rats were investigated. Tissue from different BAT depots from fed and 24 h starved rats were collected, pooled and three mitochondrial subpopulations were isolated by differential centrifugation; the M1 fraction (1000 g), the M3 fraction (3000 g) and the M15 fraction (15,000 g). Thermogenic parameters were measured in the three mitochondrial subtypes, and uncoupling protein (UCP) mRNA was determined in BAT. The results showed that starvation induced a decrease in mitochondrial turnover in BAT from both lean and obese rats. Moreover, a selective net loss of UCP from the lightest mitochondrial fraction (M15) in lean rats, with a concomitant reduction of UCP mRNA was observed. The reductions did not occur in obese rats and, as a result, a change in UCP distribution between the mitochondrial subpopulations was produced, with an increase in the M1 mitochondrial subtype. The lack of response of UCP to starvation observed in BAT of obese rats compared with the decrease seen in lean animals, is a consequence of a different mitochondrial subpopulation composition and/or a different response of a particular subpopulation to starvation.