We have tested hypotheses relating lipoprotein structure to function as measured by the relative ability to neutralize endotoxin by comparing natural human lipoproteins, a chemically defined form of reconstituted high-density lipoprotein (R-HDL), and a lipid emulsion (Intralipid). The human whole-blood system was used as an in vitro model of lipopolysaccharide (LPS) binding protein and CD14-dependent activation of cytokine production. When lipoproteins were compared on the basis of protein content, R-HDL was most effective in reducing tumor necrosis factor alpha (TNF-alpha) production followed in order by very low density lipoprotein, low-density lipoprotein, Intralipid, and natural HDL. However, when these particles were compared by protein, phospholipid, cholesterol, or triglyceride content by stepwise linear regression analysis, only phospholipid was correlated to effectiveness (r2 = 0.873; P < 0.0001). Anti-CD14 monoclonal antibodies MY4 and 3C10 inhibited LPS binding protein and CD14-dependent activation of TNF-alpha production by LPS at LPS concentrations up to approximately 1.0 ng/ml. R-HDL (2 mg of protein per ml) blocked TNF-alpha production by LPS from both smooth- and rough-type gram-negative bacteria at concentrations up to 100 ng of LPS per ml but had little effect on heat-killed gram-positive Staphylococcus aureus and no effect on other LPS-independent stimuli tested. These results support our hypothesis that LPS is neutralized by binding to phospholipid on the surface of R-HDL and demonstrate that R-HDL is a potent inhibitor of the induction of TNF-alpha by LPS from both rough- and smooth-form gram-negative bacteria in whole human blood.