The intramitochondrial distribution of steroid 11β-hydroxylation and cholesterol side-chain cleavage activity in bovine adrenocortical mitochondria have been studied. Incubation of the mitochondria with phospholipase A followed by differential centrifugation yielded four fractions: supernatant (S 3), a low-speed, compact pellet (R 2), a low-speed, loosely packed pellet (“interphase”), and a high-speed pellet (K). Steroid 11β-hydroxylation and cholesterol side-chain cleavage activity could be recovered in good yield in the supernatant fraction and separated from the succinate dehydrogenase, succinate oxidase, cytochrome c oxidase and ATPase activities, and cytochrome ( a + a 3). By controlled digestion of the mitochondria, the cholesterol sidechain activity could be separated from the 11β-hydroxylation activity and these two activities were released into the supernatant fraction at different rates than were the adenylate kinase, malate dehydrogenase, and α-ketoglutarate dehydrogenase activities. It is concluded that the cholesterol side-chain cleavage activity is located in the outer membrane compartment of the bovine adrenocortical mitochondria. Electron microscopy supported this conclusion with regard to the distribution of the cholesterol side-chain cleavage system, but left unanswered the precise distribution of the steroid 11β-hydroxylase.