The bacterial component responsible for the induction of transient cold agglutinin syndrome in rabbits after intravenous injection of heat-killed Listeria monocytogenes type 4B has been purified and biologically and chemically characterized. A purified immunoglobulin M cold agglutinin was prepared from hightiter sera resulting from the immunization of rabbits with heat-killed L. monocytogenes type 4B and was subsequently used to monitor the purification of the bacterial component responsible for its induction. The bacterial component was isolated from a hot phenol-water extract of lyophilized L. monocytogenes type 4B by multiple molecular sieve chromatography. Upon chemical analysis the purified material was found to be strikingly similar in chemical composition to gram-negative lipopolysaccharide endotoxins. The material contained 15% total fatty acid (of which 50% was fB-hydroxymyristic acid), 40 to 45% neutral sugar (glucose, galactose, and rhamnose), 11.5% amino sugar, 12% uronic acid, 2.5% 2keto-3-deoxyoctonic acid, 2% heptose, 0.87% phosphorus, and 1.6% amino acid, thereby accounting for 85 to 90% of the weight of the component. Electron micrographs of the purified material were similar to those of lipopolysaccharide preparations from gram-negative organisms. The purified material exists in aqueous solutions as large aggregates, but can be dissociated into a single smaller subunit (3.1S) by dialysis against sodium dodecyl sulfate buffer. The listerial component was toxic and pyrogenic to rabbits, producing symptoms typical of gram-negative endotoxins. Activity in the limulus lysate gelation assay and in the carbocyanine dye assay provides a further link of this material with classical gram-negative endotoxins.The clinical significance of Listeria monocytogenes, a gram-positive diphtheroid-like rod of the family Corynebacteriaceae, is becoming increasingly recognized (13). Infection in humans may result in encephalitis, meningitis, septicemia, and abortion. A peculiar result of some septicemic listeric infections in humans is the induction of a transient cold agglutinin syndrome in which antibodies are induced that can react with the host's own erythrocytes under appropriate conditions, causing a complementmediated in vivo lysis (17). The antibodies responsible for this phenomenon are of the immunoglobulin M (IgM) class and appear to be directed against the I blood group system (5).Costea and co-workers (5) developed an animal model of this response by the intravenous injection of heat-killed L. monocytogenes serotype 4B (HKLM) into rabbits. The cold agglutinin syndrome so produced was found to mimic that which occurs in humans in symptomology and pathology and was caused by IgM antibod-ies. Their results further indicated that this IgM antibody was not only induced by and directed against the HKLM, but was also able to crossreact with the erythrocytes of the host as well as other xenogenic erythrocytes which possess I blood group antigen(s). Costea et al. (7) were subsequently able to show that a crude frac...