Administration of ACTH 1-24 to 3-10 days old rats produced a significant decrease in hydrolysis of beta-casomorphin-4-nitroanilide (beta-CM-4NA) in the cytosolic fraction of brain homogenate in the first three hours after injection. Corticosterone treatment did not modify the hydrolysis of the substrate. ACTH 1-24 but not ACTH 4-10, Met-enkephalin or Leuenkephalin given to the brain homogenate resulted in a dose-dependent decrease in liberation of 4NA from beta-CM-4NA. Kinetic data suggest competitive inhibition of ACTH molecule on hydrolysis of beta-CMA-4NA. The ACTH treatment, however, did not influence the hydrolysis of Pro-Gly-4NA or Pro-Pro-4NA in the brain homogenate in vitro.