Pure monoiodo ACTH-¹²⁵I was prepared that was biologically active and free of unlabeled ACTH. Extracts of adrenal cortex that contained ACTH-sensitive adenyl cyclase, bound ACTH-¹²⁵I; extracts that lacked the ACTH-sensitive cyclase did not bind ACTH-¹²⁵I. Unlabeled ACTH inhibited the binding of ACTH-¹²⁵I. Five ACTH derivatives which varied widely in biological activity were tested. All inhibited the binding of ACTH-¹²⁵I in direct proportion to their biological activity. Albumin, insulin, and four unrelated iodinated hormones were inert. The addition of excess hormone or acetic acid produced rapid dissociation of bound ACTH-¹²⁵I. This study demonstrates directly the binding of ACTH to its biologically significant site.