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While Actinobacillus actinomycetemcomitans has been associated with rapidly progressive periodontal destruction in man, the closely related Haemophilus aphrophilus has not been related to periodontal disease. This may be due to differences in composition and structure of the lipopolysaccharides (LPS) of these dental-plaque bacteria, since LPS probably exerts a series of detrimental effects on the periodontium. LPS was prepared by the phenol-water procedure from the type strains of A. actinomycetemcomitans and H. aphrophilus, purified by hexane extraction and ultracentrifugation, and analyzed with gas chromatography and gas chromatography-mass spectrometry. While the lipid content of LPS from A. actinomycetemcomitans constituted 35.4%, it was only 18.4% in H. aphrophilus: 3-hydroxytetradecanoic and tetradecanoic acids were 21.1 and 14.3% in A. actinomycetemcomitans and 10.9 and 7.5% in H. aphrophilus. There were qualitative and quantitative differences in the polysaccharide portions of their LPS. A actinomycetemcomitans contained both D-glycero-D-mannoheptose and L-glycero-D-mannoheptose (7.8 and 11.3%); H. aphrophilus contained only L-glycero-D-mannoheptose (17.4%). The rhamnose, fucose, galactose, glucose, and glucosamine/galactosamine contents in A. actinomycetemcomitans were 2.6, 5.2, 10.1, 22.4, and 5.2%, respectively; in H. aphrophilus, they were 2.1, 2.6, 19.4, 36.4, and 3.7%. Chemical differences in LPS from A. actinomycetemcomitans and H. aphrophilus may contribute to the divergence in periodontopathogenic potential of these organisms and help taxonomic differentiation.