We have previously found that proenkephalin processing is incomplete in the neonatal rat adrenal medulla and have postulated that immaturity of either the nervous input to the gland or the endocrine hypothalamus-pituitary-adrenal axis might be involved in the failure of the gland to yield free met-enkephalin. Therefore, we investigated whether cholinergic and glucocorticoid agonists may act in vivo on neonatal proenkephalin processing; reserpine, a strong activator of precursor cleavage, was also tested. Acute administration of nicotine, pilocarpine and reserpine to 24-hour-old rats increased the content of enkephalin-containing peptides (ECP) after 72 h (4-day-old rats) and activated the posttranslational processing of proenkephalin to high, intermediate and low molecular weight peptides respectively, although free met-enkephalin was not produced. Chronic treatment with nicotine and pilocarpine neither modified the concentration of ECP nor were able to induce free metenkephalin production. Chronic administration of dexamethasone increased ECP levels in the adrenal of 4-day-old rats and caused proenkephalin processing to intermediate- and low-molecular-weight products including the production of free met-enkephalin. These results indicate that only dexamethasone was able to induce the production of met-enkephalin in the adrenal of neonatal rats, suggesting an involvement of the hypothalamus-pituitary-adrenal axis in the proteolytic maturation of proenkephalin during the ontogeny of rat adrenal medulla.