In vitro, E5564 dose-dependently inhibited LPS-mediated activation of primary cultures of human myeloid cells and mouse tissue culture macrophage cell lines as well as human or animal whole blood at nanomolar concentrations as measured by production of tumor TNF-and other cytokines. E5564 also blocked the ability of Gram negative bacteria to stimulate human cytokine production in whole blood. In vivo, E5564 blocked induction of LPS-induced cytokines and LPS or bacterial-induced lethality in primed mice. E5564 was devoid of agonistic activity when tested both in vitro and in vivo and had no antagonistic activity against Gram positive-mediated cellular activation at concentrations up to 1 μM. E5564 blocked LPS-mediated activation of nuclear factor-B in TLR4/MD-2transfected cells. In a mouse macrophage cell line, activity of E5564 was independent of serum, suggesting that E5564 exerts its activity through the cell surface receptor(s) for LPS, without the need for serum LPS transfer proteins. Similar to E5531, another lipid A-like antagonist, E5564 associates with plasma lipoproteins, causing low concentrations of E5564 to be quantitatively inactivated in a dose-and time-dependent manner. However, compared with E5531, E5564 is a more potent inhibitor of cytokine generation, and higher doses retain activity for a period of time likely sufficient to permit clinical application. These results indicate that E5564 is a potent LPS antagonist and lacks agonistic activity in human and animal model systems, making it a potentially effective therapeutic agent for treatment of disease states caused by LPS. Compared with E5531, E5564 is structurally and synthetically less complex, yet seems to possess superior activity and pharmacological characteristics. Although E5531 demonstrated potent inhibition of LPS when added to blood in vitro and in vivo, activity decreased as a function of time. This reaction has been shown to be due to interaction of E5531 with plasma lipoproteins (58) (59) (60) . E5564 is an inhibitor of LPS-mediated stimulation of responsive cells in vitro and in vivo as measured by production of cytokines, as well as morbidity and mortality associated with LPS poisoning in animal models. Because E5564 is a structural analog of the lipid A portion of LPS, it is logical to hypothesize that the antagonist interacts with the same signaling components that bind to LPS such as the soluble serum proteins LBP and sCD14, as well as membrane-associated CD14 and perhaps the TLR4/MD-2 receptor complex. E5564 blocked LPS/ sCD14-induced reporter activity in TLR4/MD-2-expressing HEK293 (61), but not TLR2-mediated signaling by heatkilled S. aureus. These findings indicate that E5564 selectively inhibits LPS signaling via TLR4/MD-2. However, a limitation to this model system is that LPS requires the presence of sCD14 for cellular activation, making it difficult to determine whether E5564 blocks LPS binding to sCD14 or TLR4/MD-2.