Smooth lipopolysaccharide (sLPS) of Brucella abortus, which is the most immunodominant component among the antigens of B. abortus isolated, has been used for diagnosis for decades. High yields of sLPS can be prepared by a modification of the procedures of Moreno et al. (J. Bacteriol. 138:361-369, 1979). Washed B. abortus cells can be disrupted by 21 freeze-quick thaw cycles and ultrasonication to separate non-membrane-bound material; then phenol extraction is performed 3 times and the phenol fraction is washed with H2O intensively. The membrane-bound sLPS can be fractionated into 3 to 5 groups according to the extent of dialysis and centrifugation. These membrane bound sLPS fractions show marked individual differences in their precipitin profile and chemical composition. Their protein content varies from 16% to 42% as determined by dye binding test and 17 to 60% by Lowry phenol method using bovine serum albumin as the standard, which indicates that these proteins associated with LPS may play important roles in the immunochemical interactions, solubility, and the heterogeneity of B. abortus lipopolysaccharides. Compared to previously published methods, a higher yield of sLPS, ranging from 3.6% to 7.7% of dried bacteria, is obtained. Group f5A, which has a standard bell shaped curve in the precipitin assay, is one of the major fractions in all three strains (1119.3, 19, 2308). The protein free sLPS (less than 1% of Lowry reactive component) can be prepared by pronase digestion. The immunochemical reactivity remains about the same before and after this treatment. The O-chains of the major fraction (f5A) of B. abortus (Strains 2308 and 19) membrane bound smooth lipopolysaccharide (sLPS) are obtained by hydrolysis of f5A native sLPS in 1% acetic acid at 100 degrees C for 2 hours. After hydrolysis, the O-chains are separated from the lipid A protein complex by centrifugation, and from small fragments by ultrafiltration of a molecular weight cut-off (MWCO) of 1.0 x 10(3). These carbohydrate haptens can be identified by precipitin-inhibition assay and further fractionated by both membrane filtration and dialysis. The size distributions of carbohydrate haptens of the endotoxins (f5A) ranged from several oligosaccharides up to 1.0 x 10(4) MWCO. Three major fractions of MWCO 8.0-10.0 x 10(3), 3.5-5.0 x 10(3), and less than 1.0 x 10(3) for both strains 2308 and 19 contain more than 85% of the total immunreactive materials.(ABSTRACT TRUNCATED AT 400 WORDS)