Rat brown adipose tissue mitochondrial subpopulations—isolated by differential centrifugation at 1000, 3000 and 8000g, giving the heavy, medium and light mitochondria—were characterized. Thus, contamination by non-mitochondrial subcellular components, morphological features, respiratory chain and antioxidant enzyme activities, both uncoupling protein 1 and mitochondrial protein content, mitochondrial DNA levels and mitochondrial integrity were measured. Results indicate that mitochondrial fractions showed important differences in the morphological, thermogenic and antioxidant properties. All the parameters studied were always higher in heavy mitochondria, which is indicative of a greater mitochondrial differentiation state.