In previous studies, enhanced toxicity of hemoglobin when combined with endotoxin has suggested a specific binding site. This binding may take place even when endotoxin-free hemoglobin is administered, due to some low levels of endotoxin usually present in the portal vein. To test this hypothesis, we administered endotoxin-free, stroma-free human hemoglobin (SFH) to rabbits in doses shown previously to be hepatotoxic or lethal. A second group of rabbits received the anti-endotoxin antibiotic polymyxin B (PB) to inactivate endotoxin, followed by a dose of SFH. A control group received intravenous PB alone. There was no differences in the incidence of hepatotoxicity among these three groups. To show the binding interactions of endotoxin with PB and endotoxin with hemoglobin, an experiment using toluidine blue was performed. According to this study, specific binding of hemoglobin with endotoxin did not occur. Our results do not agree with previous suggestions that hemoglobin-mediated hepatotoxicity is due to the interaction in vivo of hemoglobin with low levels of endogenous endotoxin.