Intraperitoneal injection of caffeine (12.5-100 mg/kg) into rats caused a significant, dose-related increase in plasma corticosterone 2 h later, when the greatest response was measured. The corticosterone response to laparotomy stress or i.v. injection of ACTH(1-24) was unaffected by prior injection of caffeine. The response to stress or caffeine was unaffected by adrenal enucleation 28 days previously. In vitro, 10 mmol caffeine/l stimulated basal release of corticosterone from adrenal quarters and potentiated the response to a sub-maximal stimulatory concentration of cyclic AMP (cAMP). The drug had no effect on release stimulated by a sub-maximal concentration of ACTH(1-24). Release of ACTH from pituitary fragments incubated in vitro was stimulated in a dose-related manner by caffeine (0.01-10 mmol/l), and the responses to hypothalamic extract and sub-maximal concentrations of corticotrophin-releasing factor (CRF-41) or arginine vasopressin (AVP), but not cAMP, were significantly enhanced by 10 mmol caffeine/l. Release of immunoreactive CRF-41 (but not AVP) was significantly increased by caffeine (0.01-10 mmol/l) added to hypothalami incubated in vitro. The response to injection of caffeine in vivo was completely prevented by pharmacological blockade of endogenous CRF release. Taken together, these results show that caffeine at high concentrations can stimulate directly the release of the hormones of the hypothalamo-pituitary-adrenocortical axis in vitro, but the fact that these concentrations are unlikely to be reached after administration in vivo suggests that the effect of caffeine may be mediated centrally.