Peripheral administration of lipopolysaccharide (LPS) may activate the hypothalamus-pituitary-adrenal (HPA) axis by way of both neural and humoral mechanisms. We have investigated whether biologically active endotoxin appears in the general circulation after intraperitoneal administration of LPS (5 or 100 micrograms/kg) to rats and whether this is a prerequisite for activation of this HPA axis. Within 15 min, endotoxin appeared in the general circulation, whereas elevations of plasma adrenocorticotropic hormone (ACTH), corticosterone, and interleukin (IL)-6 concentrations were not detected until 90 min after LPS injection. At this time, a marked interindividual variation was observed in plasma concentrations of endotoxin, ACTH, corticosterone, and IL-6. Elevated levels of plasma endotoxin were associated with elevated levels of ACTH, corticosterone, and IL-6. Intravenous administration of the LPS antagonist cationic antimicrobial protein 18 (5 mg/kg), which did not affect cytokine production in the peritoneal cavity, markedly reduced plasma ACTH, corticosterone, and IL-6 levels after 5 micrograms/kg LPS. Our results suggest that circulating endotoxin is required for the activation of the HPA axis. They also favor a role for circulating IL-6 in this response.