Using monoclonal antibodies directed against defined epitopes of endotoxin core, this study demonstrated that the presentation of lipopolysaccharide (LPS) to antibodies is critical for measuring the specific binding of antibodies to LPS structures. False cross-reactive reactions apparently were observed when free core LPS or lipid A were used as antigens in ELISA, whereas coating with complexes of high-density lipoproteins with core LPS increased both the sensitivity and the specificity of the test compared with coating with free core LPS, so that nonspecific binding of antibodies was largely avoided. Using this technique, it was not possible to find broadly cross-reactive core LPS antibodies after immunization of rabbits and humans with rough mutants of gram-negative bacteria. These observations underscore the need for careful evaluation of the potential for cross-reactivity of antisera and of monoclonal antibodies directed against endotoxin core.