Abstract Lipid X (2,3-diacyglucosamine-l-phosphate) accumulates in mutants of Escherichia coli incapable of assembling the disaccharide backbone of lipid A, the principle endotoxic moiety of bacterial lipopolysaccharide (LPS). We compared the effects of lipid X on cultured bovine aortic endothelial cell (BEC) viability and prostacyclin (PGI 2) release with those of lipid A and LPS. At 10 −5 M, both LPS and lipid A produced significant BEC cytotoxicity (percentage cytotoxicity 69 ± 4 for LPS and 51 ± 11 for lipid A) and induced a variable but consistent increase in the release of PGI 2 (11- to 73-fold increase for LPS and 4- to 6-fold increase for lipid A). Lipid X, in contrast, was not toxic and did not induce PGI 2 release at 10 −4 M. Pretreatment and coincubation of BEC with lipid X, at a concentration 100 times greater than LPS, failed to prevent LPS-mediated cytotoxicity. Intravenous infusion of lipid X in goats had no effect except for a modest elevation in the pulmonary artery pressure during the period of infusion. Moreover, pretreatment of goats with lipid X (70 μg/kg) did not block the effects of a subsequent infusion of LPS (5 μg/kg). These data suggest that a fatty acid-substituted disaccharide is the miminal molecular requirement for the numerous effects in vivo and activity in vitro induced by LPS. Furthermore, these effects are not prevented by pretreatment with a monosaccharide precursor of lipopolysaccharide, lipid X, at a dose 10- to 100-fold greater than that of LPS.