We previously reported that the serotype b antigen of Actinobacillus actinomycetemcomitans is a constituent of the polysaccharide region of lipopolysaccharide (LPS) and contains significant amount of the neutral sugars rhamnose and fucose (M. Wilson and R. Schifferle, Infect. Immun. 59:1544-1551, 1991). In the present study, we determined the structure of the O antigen of A. actinomycetemcomitans Y4 (serotype b) LPS. Aqueous phase LPS was obtained from a phenol-water extract of A. actinomycetemcomitans Y4. This material was found to react with rabbit polyclonal antiserum to serotype b but not with antisera specific for other A. actinomycetemcomitans serotypes. Analyses revealed that the O polysaccharide of Y4 LPS consists of a polymer of trisaccharide repeating units composed of D-Fuc, AL-Rha, and D-GalNAc residues. An identical structure was obtained for the O polysaccharide of LPS from A. actinomycetemcomitans JP2, another serotype b strain. These results indicate that the serotype b antigen of A. actinomycetemcomitans is defined by a trisaccharide repeating unit present in the O polysaccharide of LPS.