An enzyme-linked immunosorbent assay for antibody to the lipopolysaccharides (LPSs) of a rough strain of Escherichia coli (J5) and Neisseria gonorrhoeae was developed. When standard methods of this assay were applied to these LPSs, no antibody was measured. By radiolabeling smooth and rough endotoxins with 51Cr, it was discovered that the rough LPS was not adhering to the polystyrene tubes. When Mg++ was added, however, all endotoxins absorbed to the solid phase. With this assay antiserum to core glycolipid had higher titers against its smooth parent strain than did homologous glycolipid had higher titers against its smooth parent strain that did homologous antiserum, and antisera to gonococci had high homologous but low heterologous titers. Thus, these data demonstrate that bulky O side chains do not hinder the penetration of antibody to core glycolipid and that LPSs from gonococci are heterogeneous but do cross-react.