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Recent biochemical studies on the functional zonation of the human adrenal cortex suggest that one of the earlier principal effects of corticotrophin (ACTH) on steroid biosynthesis occurs at the fasciculo-reticular border. This report deals with procedures for the study of the 11β-hydroxylase activity in microgram amounts of adrenal tissue and with the distribution of this activity throughout the adrenal cortex of the rat. ACTH administration is shown to stimulate the 11β-hydroxylase activity at the fasciculo-reticular border.