In an attempt to obtain pure and well characterized smooth lipopolysaccharide (S-LPS) and rough lipopolysaccharide (R-LPS), smooth and rough strains of Brucella abortus were extracted by two different modifications Qf the phenolwater method. S-LPS was obtained in the phenol phase, and R-LPS was obtained in the aqueous phase. Further purification was accomplished by treatment with enzymes, detergents, NaI as a chaotropic agent to separate non-covalently bound contaminants, and by gel filtration. The degree of purity of the molecules was determined by chemical and immunological analysis and by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. Lipid identification by gas-liquid chromatography showed seven major fatty acids. Palmitic acid accounts for about 50%, stearic acid accounts for about 10%, and hydroxylated fatty acids account for less than 5% of total fatty acids. 2-Keto-3-deoxyoctonate but not heptose was detected in the sugar analysis. Protein was found to be firmly bound to S-LPS but not to R-LPS. Several investigators have shown that the crude endotoxin from smooth Brucella spp. is isolated from the phenol phase when cells are extracted with hot phenol-water (2, 3, 6, 18, 22, 23, 30). This fraction, containing 2-keto-3-deoxyoctonate (KDO), sugars, and lipids, presents both similarities and differences when compared with lipopolysaccharides (LPS) from Enterobacteriaceae. Examples of such similarities and differences include observations on toxicity, pyrogenicity, and hybrid formation with Escherichia coli LPS. Baker and Wilson (1, 2) found that endotoxin preparations of smooth Brucella abortus were toxic for mice, but were less toxic than those from E. coli. Leong et al. (30) described qualitative differences in biological activity but not structural differences between brucella endotoxins and enterobacterial endotoxins. Jones et al. (22) reported that saline-extracted rough LPS (R-LPS) from B. ovis was toxic for mice and had limulus lysate gelation activity (LLGA) comparable to that of E. coli LPS. Munoz et al. (36) showed that an endotoxin preparation of smooth B. abortus had comparable LLGA to LPS from several different strains of Salmonella and E. coli, but did not increase sensitivity of mice to histamine. Because these experiments were performed with crude brucelia endotoxin preparations rather than with purified LPS, it was not possible to establish any relationship between chemical composition and biological activity that could help to explain the differences in behavior of these LPS molecules. The work reported here describes preparation and analytical data for purified smooth LPS (S-LPS) and R-LPS from B. abortus which is essential to experiments on biological activity. These, in turn, may contribute to the understanding of the relationship of chemical composition to activity. MATERIALS AND METHODS Bacterial cultures. The bacterial strains used, their characteristics, and the conditions of culture were described previously (22). Extraction of crude LPS. The crude S-LPS (fM) was extr...