Brown adipose tissue mitochondria from cold-stressed guinea pigs isolated in sucrose-HEPES-EDTA medium (HEPES = N-2-hydroxyethylpiperazine- N′-2-ethanesulphonic acid) possess several criteria of loose coupling (J. I. Pedersen and H. J. Grav, Eur. J. Biochem., 25 (1972) 75), and the present studies on the steady-state oxidation-reduction level of the cytochrome b complex under various experimental conditions have confirmed this conclusion. Generally, the kinetics as well as the steady-state redox level of b-type cytochromes in brown adipose tissue mitochondria differ markedly from those of coupled liver mitochondria of the same animals. The main features of these mitochondria are: 1. 1. In the presence of rotenone, cyanide, ascorbate, and N, N, N′, N′-tetramethyl- p-phenylenediamine (TMPD) the addition of carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) induces no oxidation of b-type cytochromes in brown adipose tissue mitochondria whereas a 30% oxidation was observed with liver mitochondria. This fact indicates that freshly isolated brown adipose tissue mitochondria are completely deenergized. 2. 2. The percentage degree of reduction of b 561 by succinate increases almost linearly from pH 6.0 (approx. 10%) to pH 8.0 (approx. 32%) in brown adipose tissue mitochondria, whereas the reduction of b 561 in liver mitochondria (approx. 16%) is almost independent of pH in the same region. 3. 3. With brown adipose tissue mitochondria, but not with liver mitochondria, the subsequent reduction of b 565 by ATP was dependent on added P i. 4. 4. The ATP-induced reduction of b 565 in liver mitochondria was completely inhibited by oligomycin whereas 87% inhibition was observed with brown adipose tissue mitochondria. The uncoupler FCCP, however, completely inhibited the ATP response. The oligomycin-insensitive reduction could also be induced by other highenergy nucleotides such as GDP and GTP. 5. 5. The ATP-induced reduction of b 565 in brown adipose tissue mitochondria revealed a pH dependence very different from that of liver mitochondria; the pH optium was around 7.4 in liver mitochondria whereas that of brown adipose tissue mitochondria was approx. 1 pH unit lower. 6. 6. The concentration of ATP needed for half-maximal reduction of b 565 was 30 times higher with brown adipose tissue mitochondria than with liver mitochondria under identical experimental conditions. 7. 7. The significance of the pH and the intramitochondrial concentrations of adenine nucleotides and phosphate in the regulation of the coupling of respiration to phosphorylation in brown adipose tissue mitochondria is discussed.