The injection of repeated doses of lipopolysaccharide (LPS) results in attenuation of the febrile response, which is called endotoxin tolerance. We tested the hypothesis that NO arising from inducible nitric oxide synthase (iNOS) plays a role in endotoxin tolerance, using not only pharmacological trials but also genetically engineered mice. Body temperature (Tb) was measured by biotelemetry in mice treated with N^G-monomethyl-L-arginine (L-NMMA, 40 mg/kg; a non-selective NOS inhibitor) or aminoguanidine (AG, 10mg/kg; a selective iNOS inhibitor) and in deficient iNOS gene (iNOS KO) mice. Tolerance to LPS was induced by means of three consecutive LPS (100 µg/kg) intraperitoneal injection (i.p.) at 24h intervals. In wild-type mice, we observed a significant reduction of the febrile response to repeated administration of LPS. Injection of L-NMMA and AG markedly enhanced the febrile response to LPS in tolerant animals. Conversely, iNOS-KO mice repeatedly injected with LPS did not become tolerant to the pyrogenic effect of LPS. These data are consistent with the notion that nitric oxide modulates LPS tolerance in mice and that iNOS isoform is involved in NO synthesis during LPS tolerance.