Bactericidal/permeability-increasing protein (BPI), a cationic protein found in neutrophil granules, binds with high affinity to gram-negative bacterial lipopolysaccharide (LPS) and can inhibit its actions in vitro. The in vivo efficacy of a recombinant 23-kDa amino-terminal LPS-binding fragment of BPI (rBPI23) was assessed in a mouse model of lethal endotoxemia. Systemic administration of rBPI23 protected actinomycin D-sensitized mice from lethal LPS (Escherichia coli O111:B4) challenge in a dose-dependent manner, with almost complete protection at the highest dose (10 mg/kg; 93% survival vs. 13% in vehicle-treated controls). Surviving rBPI23-treated animals did not show histopathologic signs of tissue damage evident in control animals that had died after LPS challenge. rBPI23 also attenuated the LPS-induced elevation in serum levels of tumor necrosis factor-alpha and interleukin-1 alpha, mediators believed to be involved in the pathogenesis of endotoxemia and sepsis. Thus, rBPI23 may be a potential new therapeutic agent for the treatment of gram-negative bacterial infection and sepsis.