This work examines the role of nitric oxide (NO) in the periphery (i.e., on the pituitary) and the brain (particularly on corticotropin-releasing factor [CRF] and vasopressin [VP] neurons in the paraventricular nucleus [PVN] of the hypothalamus) as a modulator of the ACTH response to lipopolysaccharide. We previously showed that NO restricted the pituitary response to VP while it facilitated the synthesis of PVN CRF and VP. In our experience, only relatively high doses of lipopolysaccharide (>50 µg/kg, injected intravenously [iv]) cause detectable increases in PVN neuronal activation. Our hypothesis, therefore, was that pituitary NO-VP interactions would predominate in rats injected with a low dose of lipopolysaccharide (0.5 µg/kg, iv) while the stimulatory influence of the gas on PVN neuronal activity would play an important role following iv injection of a large dose of lipopolysaccharide (50 µg/kg, iv). We observed that the ability of 0.5 µg/kg lipopolysaccharide to release ACTH was significantly enhanced by the subcutaneous (sc), but not the intracerebroventricular (icv) injection of L-NAME, an arginine derivative that blocks NO synthesis. The effect of sc L-NAME was reversed by immunoneutralization of endogenous VP, which indicated that in this model, the ability of lipopolysaccharide to release ACTH depended, at least in part, on the influence exerted by NO on the pituitary response to VP. In rats injected with the high lipopolysaccharide dose, the sc injection of L-NAME decreased plasma ACTH levels compared to those in rats pretreated with the vehicle. The effect of sc L-NAME was not significantly altered by VP antibodies. These results indicate that in this model, the primary influence of NO was exerted in the PVN and/or its afferents and that it did not depend on a peripheral, VP-mediated effect of the gas. On the one hand, these data are at odds with our finding that the icv injection of L-NAME only marginally altered the ACTH response to the large dose of lipopolysaccharide. As icv injected L-NAME should have primarily decreased hypothalamic, but not pituitary NOS, its only modest influence on ACTH release may have been due to a balance between stimulating and inhibiting effects of NO within the brain. As high doses of lipopolysaccharide increase brain levels of prostaglandin, monoamine, and proinflammatory cytokines, it will be important to investigate the influence exerted by NO on these secretagogues and on their interactions with PVN CRF and VP neurons, which may help us resolve the issues raised by our results. Collectively, these data support our hypothesis that the mechanisms mediating the ACTH response to a low lipopolysaccharide concentration involve the inhibitory VP-mediated influence of NO on pituitary activity. By contrast, the stimulatory effect of high doses of lipopolysaccharide on ACTH release depends, at least in part, on the ability of NO to upregulate PVN neuronal activity.