Salmonella madelia reported to express the O-antigenic factors 1, 6, 14, and 25, defined in the Kauffmann–White classification system, was found to produce three different homogeneous lipopolysaccharides, which differed in having three structurally distinct O-polysaccharide components. The O-polysaccharide fraction obtained by mild acetic acid hydrolysis of the S. madelia lipopolysaccharide was analyzed by chemical composition, nitrous acid deamination, periodate oxidation, methylation, and 1H and 13C nuclear magnetic resonance methods and was demonstrated to be composed of three polysaccharides, PS(I), PS(II), and PS(III), which had the structures of repeating oligosaccharide units:[Formula: see text]Key words: Salmonella madelia, lipopolysaccharide, structure, analysis, nuclear magnetic resonance.