To test whether the diurnal rhythm in stress responsiveness is dependent on corticosterone (B)-mediated negative feedback, the responses of intact (SHAM) and adrenalectomized (ADX) rats to restraint for 3-90 minutes or ip injection with saline in the morning (AM) and the evening (PM) were compared. In both SHAM and ADX rats, ACTH responses to restraint stress were larger in the AM. In intact rats, this could have resulted from both fast negative feedback, due to the rate of rise of B during the stress in the PM, and delayed negative feedback, due to the high basal concentrations of B before the stress in the PM. However, this diurnal pattern of stress responsiveness was not dependent on B, as the same relative responses to restraint and ip injection were found in ADX rats. To determine whether the lack of response of ADX rats in the PM to stress was due to a loss of sensitivity to endogenous secretagogues, ADX rats were given CRF + arginine vasopressin (AVP) while anesthetized with ether after 30 min of restraint. In both the AM and the PM, the pituitaries were able to respond to exogenous secretagogues. A second novel finding was that in the PM, but not the AM, plasma ACTH concentrations in the ADX rats decreased substantially during the period of restraint, despite the lack of B-mediated negative feedback. In the AM and the PM, ADX rats were restrained for 30 min and then stressed with ether for 6 min. The ACTH concentrations were not different before and after ether, suggesting that, although the pituitaries of ADX rats are able to respond to exogenous CRF + AVP after stress, an additional stress of ether exposure no longer stimulates endogenous CRF and AVP release after 30 min of restraint at either time of day. After 90 min of restraint in the AM and the PM, the relationship between ACTH and B was positive, not negative, providing no evidence of ongoing B-mediated negative feedback in the SHAM rats. Therefore, the same mechanism responsible for the decrease in ACTH secretion in ADX rats may occur in SHAM rats as well. From these results, we conclude that the diurnal rhythm in stress responsiveness and, in the PM in the ADX rats, the decrease in plasma ACTH during stress, are largely independent of B.