A lipopolysaccharide (LPS)-binding lectin was recovered from the serum of Limulus polyphemus by ion-exchange chromatography. Electrophoretic analysis of this lectin preparation revealed three poorly migrating bands. When whole serum was incubated with glycolipid obtained from the Rc mutant of Salmonella minnesota prior to electrophoresis, bands corresponding to those seen in the partially purified lectin were missing, suggesting that the recovered material was composed of isolectins. Qualitative precipitin tests revealed no reactivity of this purified lectin with lipid A fractions or with LPS devoid of 2-keto-3-deoxyoctonate (KDO). The agglutination of chicken erythrocytes by this lectin was inhibited by both N-acetyl-neuraminic acid and KDO. Erythrocytes complexed with glycolipid from the Re mutant of S. minnesota were strongly agglutinated by this lectin. We conclude that this LPS-binding lectin is specific for the KDO portion of the molecule and that it is identical to the previously described sialic acid-binding lectin from L. polyphemus. This lectin may play a role in the host defense mechanisms of Limulus.