Serotonin (5-HT) modulates the hypothalamic-pituitary-adrenal (HPA) axis response to stress. We examined the effect of chronic restraint stress (CRS; 20 min/day) as compared to control (CTRL) conditions for 14 days, on: 1) restraint-induced ACTH and corticosterone (CORT) secretion in rats pretreated with vehicle or SB-656104 (a 5-HT7 receptor antagonist); 2) 5-HT7 receptor-like immunoreactivity (5-HT7-LI) and protein in the hypothalamic paraventricular nucleus (PVN) and adrenal glands (AG); 3) baseline levels of 5-HT and 5-hydroxyindolacetic acid (5-HIAA), and 5-HIAA/5-HT ratio in PVN and AG; and 4) 5-HT-like immunoreactivity (5-HT-LI) in AG and tryptophan hydroxylase (TPH) protein in PVN and AG. On day 15, animals were subdivided into Treatment and No treatment groups. Treatment animals received an i.p. injection of vehicle or SB-656104; No Treatment animals received no injection. Sixty min later, Treatment animals were either decapitated with no further stress (0 min) or submitted to acute restraint (10, 30, 60 or 120 min); hormone serum levels were measured. No Treatment animals were employed for the rest of measurements. CRS decreased body weight gain and increased adrenal weight. In CTRL animals, acute restraint increased ACTH and CORT secretion in a time of restraint-dependent manner; both responses were inhibited by SB-656104. Exposure to CRS abolished ACTH but magnified CORT responses to restraint as compared to CTRL conditions; SB-656104 had no effect on ACTH levels but significantly inhibited sensitized CORT responses. In CTRL animals, 5-HT7-LI was detected in magnocellular and parvocellular subdivisions of PVN and sparsely in adrenal cortex. Exposure to CRS decreased 5-HT7-LI and protein in the PVN, but increased 5-HT7-LI in the adrenal cortex and protein in whole AG. Higher 5-HT and 5-HIAA levels were detected in PVN and AG from CRS animals but 5-HIAA/5-HT ratio increased in AG only. Finally, whereas 5-HT-LI was sparsely observed in the adrenal cortex of CTRL animals, it strongly increased in the adrenal cortex of CRS animals. No TPH protein was detected in AG from both animal groups. Results suggest that CRS promotes endocrine disruption involving decreased ACTH and sensitized CORT responses to acute restraint. This phenomenon may be associated with increased function and expression of 5-HT7 receptors as well as 5-HT turnover in AG.