1. Hog adrenocortical mitochondria, of which purity was assured by electron microscopic observation and by determination of the microsomal steroid 21-hydroxylase [EC 1.14.1. 8], were fractionated by centrifugation after sonication. The precipitates obtained between 9, 000 and 105, 000×g, and between 105, 000 and 150, 000×g as well as the 150, 000×g supernatant of the sonicated mitochondria were examined with respect to their enzymatic activities and cytochrome contents. 2. The 105, 000Xg precipitate was identified as the inner membrane preparation, the 150, 000×g precipitate as the outer membrane preparation, and the 150, 000×g supernatant as the matrix preparation, respectively, using succinate dehydrogenase [EC 1.3.99.1] as the marker for inner membrane, the Amytal-insensitive NADH cytochrome c reductase and cytochrome bs for outer membrane, and malate dehydrogenase [EC 1.1.1. 37] and malic enzyme [EC 1.1.1. 40] for matrix. 3. Incubation for the assay of hydroxylases of cholesterol side chain-cleaving enzyme system, in the presence or absence of exogenous NADPH diaphorase [EC 1.6. 99.1] and adrenodoxin, clearly demonstrated that these enzyme activities were located in the inner membrane. 4. Acetone treatment of mitochondria was not effective for the differential extraction of mitochondrial enzymes. 5. Present results together with the previous ones seem to establish the localization of the P-450 and hydroxylases of the cholesterol side chain-cleaving enzyme system at the inner mitochondrial membrane of the hog adrenal cortex.