Daytime restriction of food availability phase-shifts the diurnal periodicity of plasma corticosterone (B) concentration in rats. However, the changes at the various functional levels in the hypothalamic-pituitary-adrenal (HPA) axis are not completely understood. We performed studies in two groups of Wistar rats, a control group with free access to food and a food-restricted group with access to food only from 09.00 to 11.00 h for 14 days. The basal plasma B and ACTH concentrations in the control group presented diurnal variation with higher values in the afternoon than in the morning. In the food-restricted group there was a 12-hour shift of peak B values. Plasma ACTH levels were high in the morning, but there was no significant difference between morning and afternoon values. Plasma AVP presented no diurnal variation in the control group. In the food-restricted group, the diurnal variations of plasma AVP and ACTH-B did not coincide. There was no detectable nycthemeral variation of pituitary ACTH or hypothalamic CRH and AVP contents in either group. Plasma ACTH did not exhibit any diurnal variation in response to CRH in free-fed or food-restricted rats anesthetized with chlorpromazine-morphine-pentobarbital. The administration of ACTH (1-24) resulted in higher B levels in the afternoon in control rats pretreated with dexamethasone once or twice. In the food-restricted group there was a 12-hour shift in the adrenal response to ACTH (1-24). These patterns of response were abolished in both groups when the rats were anesthetized. An in vitro study showed no diurnal variation in adrenal responsiveness to 10-6M-10-9M ACTH (1-24) stimuli. Our results suggest that anesthesia-related neural or humoral factor(s) other than ACTH are involved in the diurnal periodicity of the HPA axis in free-fed rats and in its shifts in food-restricted rats.