We studied the long-term effect of neonatal treatment with the synthetic glucocorticoid dexamethasone (DEX) on stress responsivity later in life. It was found that the plasma adrenocorticotropin hormone (ACTH) and corticosterone (CORT) responses induced by novelty or conditioned fear stress were markedly attenuated in adult rats that had been neonatally treated with DEX as compared with saline (SAL)-treated controls. Since there were no differences in the heart rate, body temperature, plasma noradrenaline, plasma adrenaline and behavioral responses to these stressors, this points to a deficit within the hypothalamic-pituitary-adrenal (HPA) axis of DEX rats. We found no differences between DEX and SAL rats in basal plasma CORT concentrations measured throughout the circadian cycle, nor in the fraction unbound of CORT circulating under resting conditions, indicating normal tonic regulation of the HPA axis in DEX rats. Since we also found no differences in the hormonal responses induced by intravenous injection of graded doses of ACTH or corticotropin-releasing hormone (CRH), we investigated the sensitivity of the HPA response to stress for inhibition by glucocorticoids. Pretreatment with a low dose of CORT that did not affect the HPA response of SAL rats markedly inhibited the ACTH and CORT responses induced by novelty stress in DEX rats. This strongly suggests that an enhanced corticosteroid feedback underlies the blunted HPA response to stress in DEX rats. Finally, using quantitative immunocytochemistry, we found an increase in arginine-vasopressin (AVP) but not CRH stores in the external zone of the median eminence, suggesting an altered AVP/CRH ratio in the secretory output of the hypophysiotropic paraventricular nucleus. Taken together, our results show that exposure to DEX during early life leads to hyporesponsivity of the HPA axis to stress most likely due to hypersensitivity of the axis for negative feedback by corticosteroids at the suprapituitary level.