Electron transport pathways in bovine heart mitochondria were investigated by adding inhibitors to electron transport reaction systems. After addition of the inhibitors rotenone and mersalyl, reaction rates were 0.024 and 0.056, respectively, in terms of ΔA_(550㎚). These rates corresponded to 84.4% and 63.6% inhibition compared with a 0.154 absorption change per minute for a reaction system with no inhibition. No reaction was observed with the addition of both inhibitors. No change was observed when the reaction system of the outer mitochondrial membrane was exposed to rotenone, while complete inhibition occurred when mersalyl was added to the system. It was concluded that an electron transport region which is insensitive to rotenone but is sensitive to mersalyl exists in the outer membrane. Protein solubilization was investigated by treating the outer mitochondrial membrane for 15 h with the surfactant n-dodecyl-(imaltoside (DMS) and digitonin in a ratio of protein to surfactant of 2:1. NADH-cytochrome bs reductase (NCBR) solubilization by DMS was observed but digitonin had no effect. This result shows that DMS is a better detergent than digitonin for NCBR extrnction from the outer membrane of mitochondria. NCBR was purified by chromatography using DEAF-cellulose resin and Bio-gel for DMS extraction from the outer membrane. The purity of the enzyme was verified and the molecular weight was 39,000∼40,000 daltons based on electrophoreses. The Michaelis constants for substrates NADH, NADPH, ferricyanide for NCBR were 0.043 mM, 0.076 mM, and 0.23 mM respectively with respective maximum velocities of 0.39, 0.36, and 0.37 in terms of an absorption change at 420 ㎚ per minute. These values indicate that the substrate affinity of NCBR is greatest for the normal reactant, NADH.