Recent data suggest that heat shock protein-70 (HSP-70), an intracellular protein, can exist in the extracellular compartment and signal through the CD14/TLR4 pathway. In this study, we tested the hypothesis that extracellular HSP-70 induces endotoxin (LPS) tolerance. Using human monocyte cell line (THP-1), initial dose-response experiments were conducted to determine a subthreshold concentration of HSP-70 that does not induce NF-κB activity. Differentiated THP-1 cells were preconditioned with subthreshold concentration (0.03 μg/ml HSP-70) for 18 h, followed by LPS stimulation (1 μg/ml) for 4 h. Preconditioning with HSP-70 decreased subsequent LPS-mediated NF-κB-dependent promoter activity and was accompanied by significant decreases of supernatant TNF levels. Furthermore, human monocytes isolated from human volunteers, subsequently preconditioned with HSP-70, demonstrated LPS tolerance as evidenced by abrogated supernatant TNF levels. Additional experiments were conducted to exclude the possibility of endotoxin contamination of HSP-70 by boiling HSP-70 at 100 C for 1 h or preconditioning with equivalent concentrations of endotoxin as present in the HSP-70 preparation. These experiments indicated that induction of tolerance was not secondary to endotoxin contamination. Neutralization experiments with an anti-HSP-70 Ab confirmed the specificity of HSP-70 in tolerance induction. Preconditioning with HSP-70 attenuated cytosolic degradation of inhibitor κB-α and inhibited activation of inhibitor κB kinase following LPS stimulation. HSP-70 preconditioning decreased phosphorylation of the p65 subunit of NF-κB following LPS stimulation. These data suggest a novel role for extracellular HSP-70 in modifying mononuclear cell responses to subsequent LPS challenge.