The structures of lipids A isolated from the lipopolysaccharides (LPSs; endotoxins) of three different pathogenic Bordetella bronchiseptica strains were investigated by chemical composition and methylation analysis, gas chromatography-mass spectrometry, nuclear magnetic resonance, and plasma desorption mass spectrometry (PDMS). The analyses revealed that the LPSs contain the classical lipid A bisphosphorylated beta-(1-->6)-linked D-glucosamine disaccharide with hydroxytetradecanoic acid in amide linkages. Their structures differ from that of the lipid A of Bordetella pertussis endotoxin by the replacement of hydroxydecanoic acid on the C-3 position with hydroxydodecanoic acid or dodecanoic acid and the presence of variable amounts of hexadecanoic acid. The dodecanoic acid is the first nonhydroxylated fatty acid to be found directly linked to a lipid A glucosamine. The lipids A were heterogeneous and composed of one to three major and several minor molecular species. The fatty acids in ester linkage were localized by PDMS of chemically modified lipids A. B. pertussis lipids A are usually hypoacylated with respect to those of enterobacterial lipids A. However, one of the three B. bronchiseptica strains had a major hexaacylated molecular species. C-4 and C-6' hydroxyl groups of the backbone disaccharide were unsubstituted, the latter being the proposed attachment site of the polysaccharide. The structural variability seen in these three lipids A was unusual for a single species and may have consequences for the pathogenicity of this Bordetella species.