ACTH was prepared by different procedures and incorporated into a partially hydrolyzed gelatin vehicle. A gelatin medium enhances the effect of all prepns. tested when given subcut. The prepns. were standardized by the provisional U.S.P. intraven. assay procedure using the adrenal ascorbic acid depletion method in hypophysectomized rats. Some standardized prepns. differed in potency by about 300% when injected subcut. in humans or in rat assay. Correlation between animal and human bio-assay of corticotropin appears dependent upon use of the same mode of admn. in both. Young et al. (1951). Dixon et al. (1951), and Moyer et al. (1952) postulated the existence of multiple ACTH hormones as indicated by 2 different assay methods utilizing 2 different indices of response. Evidence indicates that by varying the route of admn. (intraven. and subcut.) and utilizing the same response index (adrenal ascorbic acid), differences in relative potency appear. The magnitude of difference can be made parallel to that utilizing a 2d response index (adrenal to wt.), following subcut. admn. This fact would tend to contradict the postulate of 2 pituitary derived ACTH hormones, one affecting adrenal ascorbic acid depletion, the 2d adrenal wt. ACTH prepns. in gelatin, standardized by the provisional U.S.P assay, were assayed by the adrenal ascorbic acid depletion method, an adrenal wt. method, and a thymus involution method, all following subcut. admn. All the above assays correlated satisfactorily with clinical findings. However, the thymus involution assay on 23-day old normal female rats appears to be the most promising.