To investigate the simultaneous effects of dexamethasone on peripheral and central adrenocorticotropic hormone (ACTH) systems, rats were treated with dexamethasone or saline for 4 days. Pituitary, plasma, hypothalamus and cerebrospinal fluid (CSF) were then collected and analyzed for ACTH immunoreactivity. Additionally, hypothalamic tissue extracts were analyzed for corticotropin-releasing hormone (CRH) immunoreactivity. Dexamethasone significantly lowered peripheral levels of ACTH as measured in pituitary and plasma. Hypothalamic ACTH content significantly increased while CSF ACTH significantly decreased with dexamethasone treatment. Hypothalamic CRH concentrations showed a small but statistically insignificant decrease. These results (1) suggest that prolonged exposure to dexamethasone affects central as well as peripheral ACTH activity, (2) corroborate our previous findings in rhesus monkeys of decreased CSF ACTH in response to prolonged dexamethasone treatment, (3) suggest that dexamethasone may inhibit the release of ACTH from hypothalamic neurons into the CSF, and (4) provide evidence that the effect of dexamethasone on pituitary ACTH content is of greater magnitude than its effect on hypothalamic CRH.