Lipoproteins isolated from normal human plasma can bind and neutralize bacterial lipopolysaccharide (LPS) and may represent an important mechanism in host defense against gram-negative septic shock. We sought to define the components of high-density lipoproteins (HDL) that are required for neutralization of LPS. To accomplish this we have studied the functional neutralization of LPS by native and reconstituted HDL using a rapid assay that measures the CD14-dependent activation of leukocyte integrins on human neutrophils. Reconstituted HDL particles (R-HDL), prepared from purified apolipoprotein A-I (apoA-I) combined with phospholipid and free cholesterol, were not sufficient to neutralize the biologic activity of LPS. However, addition of recombinant LPS binding protein (LBP), a protein known to transfer LPS to CD14 and enhance responses of cells to LPS, enabled prompt binding and neutralization of LPS by R-HDL. Thus, LBP appears capable of transferring LPS not only to CD14 but also to lipoprotein particles. These results suggest that in addition to its ability to transfer LPS to CD14, LBP may also transfer LPS to lipoproteins. Additional studies demonstrated that LBP copurifies with native apoA-I containing lipoprotein (Lp(A-I)) particles. Since LBP appears to be physically associated with lipoproteins in plasma, it is positioned to play an important role in the neutralization of LPS.