Hemoglobin (Hb) induces heme oxygenase-1 (HO-1), which catalyzes the breakdown of heme to bilirubin, and ferritin. Rats pretreated with Hb have been shown to survive lethal doses of lipopolysaccharide (LPS; see L. Otterbein, S. L. Sylvester, and A. M. Choi. Am. J. Respir. Cell Mol. Biol. 13: 595-601, 1995). The physiological basis of this increased survival and the mechanism(s) involved in the protection against LPS by Hb are unknown. Here we investigated 1) the effects of Hb on the hemodynamic and biochemical parameters of LPS-induced tissue injury and 2) the mechanism(s) by which Hb conferred protection against shock and tissue injury. Hb-treated rats maintained normal mean arterial blood pressure, whereas control rats experienced cardiovascular collapse after a lethal dose of LPS. Hepatic and renal functions, peripheral white blood cells, serum lactate dehydrogenase, and phosphate also remained normal after LPS in Hb-treated rats. Hb also attenuated LPS-induced neutrophil alveolitis and tumor necrosis factor-alpha levels. Pretreatment with both desferoxamine, which, like ferritin, can bind iron, and with exogenous apoferritin failed to protect against LPS. In contrast, treatment with Hb plus desferoxamine, which induced HO-1 but not ferritin, did protect against LPS. Treatment with iron-dextran, which induced ferritin but not HO-1, did not protect against LPS. We conclude that Hb pretreatment reduces the inflammatory and physiological consequences of LPS and that the Hb-induced protection against LPS is dependent on HO-1 and not ferritin induction.