The present study was designed to define further the relationships between ACTH and corticosterone secretion after repeated administration of a discrete restraint stress in rats. The possibility that plasma ACTH and corticosterone responses to stress may be modified by prior exposure to stress was examined in male rats using a 2-min restraint stress. The peak plasma ACTH response to a single restraint stress occurred at 2.5-5 min after the onset of the stress, and plasma ACTH returned to the basal concentration by 30 min. The plasma corticosterone concentration after this stress peaked at 15-30 min and returned to the control range by 60-90 min. The time courses of the plasma ACTH and corticosterone responses to restraint stress after administration of three prior stresses at 90-min intervals were similar to those after a single stress. Stress-induced increments in plasma concentrations of ACTH and corticosterone were similar in rats that received either a single restraint stress or as many as seven stresses repeated at 90-min intervals. Next, we examined the plasma ACTH and corticosterone responses to repeated stress applied at intervals of less than 90 min (30 or 60 min), that is, at times at which the plasma corticosterone concentration had not yet returned to basal levels. The plasma ACTH responses to the second stress were similar in magnitude and duration to the response after a single stress, whether the second stress was applied 30, 60, or 90 min after the first stress. The plasma corticosterone response to a second stress applied at 90 min was identical to the response after the initial stress. In contrast, the plasma corticosterone responses to a second stress applied 30 or 60 min after the initial stress were markedly reduced. The decrease in plasma corticosterone response to the second stress did not result from a decrease in secretion of bioactive ACTH; no difference was found between the magnitude of the plasma ACTH response to the initial stress and a subsequent stress applied 30 min later using either bioassay or immunoassay measurements. Also, the rate of corticosterone catabolism was not increased by prior stress; the rate of disappearance of corticosterone from plasma was identical after an initial or a subsequent stress applied at 30 min. We were unable to demonstrate decreased adrenocortical responsiveness to ACTH after an initial stress in dexamethasone-suppressed rats; in these rats the plasma corticosterone response to exogenous ACTH was not decreased by prior restraint stress. These data clearly define a period of decreased adrenocortical response to subsequent stress after stress-induced activation of adrenocortical secretion. Furthermore, this altered adrenocortical response appears to be mediated by a nonadrenocorticotropin mechanism.