In experimental animals and humans, bacterial endotoxin activates the hypo-thalamus-pituitary-adrenal (HPA) axis. The pathways by which endotoxin stimulates adrenocorticotropic hormone (ACTH) and corticosterone secretion are uncertain. In the present study we compared the role of hypothalamic corticotropin-releasing hormone (CRH) in the activation of the HPA axis by a low (2.5 µg/kg) and a high (2.5 mg/kg) dose of bacterial endotoxin. Two experimental models were applied using chronically cannulated male Wistar rats. In the first model, rats were subjected to lesions of the hypothalamus that interrupted dorsal, lateral and frontal input to the median eminence (anterolateral deafferentation) or to sham operation and rats were used 7 days later. Before and at hourly intervals after endotoxin (2.5 µg/kg i.p.), blood samples were taken for the determination of plasma ACTH and corticosterone concentrations. Deafferentation of the hypothalamus strongly attenuated the elevations in plasma ACTH and corticosterone concentrations by a low dose of endotoxin compared to the responses in sham-operated animals. The second model involved passive immunization to CRH using a monoclonal antibody to rat/human CRH (PFU83). PFU83 (90 nmol/rat) abolished the elevation of plasma ACTH concentrations and attenuated corticosterone responses to a low dose of endotoxin (2.5 µg/kg i.p.) compared to that in control IgG-treated rats. Since the corticosterone responses to endotoxin were less effectively inhibited by the antibody than the ACTH responses, we postulate that non-ACTH-dependent mechanisms may contribute to the corticosterone response to endotoxin. The same dose of PFU83 attenuated the endotoxin-induced ACTH responses to a high dose of endotoxin (2.5 mg/kg i.p.) by ± 45% but did not affect plasma corticosterone concentrations. We conclude that the ACTH responses to a low dose of endotoxin in rats are completely mediated by secretion of CRH from hypothalamic CRH-producing neurons that project to the median eminence, whereas the ACTH response induced by high doses of endotoxin is only partially dependent on