<jats:title>ABSTRACT</jats:title> <jats:p> <jats:italic>Porphyromonas gingivalis</jats:italic> , an anaerobic gram-negative bacterium associated with chronic periodontitis, can agglutinate human erythrocytes. In general, hemagglutination can be considered the ability to adhere to host cells; however, <jats:italic>P. gingivalis</jats:italic> -mediated hemagglutination has special significance because heme markedly accelerates growth of this bacterium. Although a number of studies have indicated that a major hemagglutinin of <jats:italic>P. gingivalis</jats:italic> is intragenically encoded by <jats:italic>rgpA</jats:italic> , <jats:italic>kgp</jats:italic> , and <jats:italic>hagA</jats:italic> , direct evidence has not been obtained. We demonstrated in this study that recombinant HGP44 <jats:sub>720-1081</jats:sub> , a fully processed HGP44 domain protein, had hemagglutinating activity but that an unprocessed form, HGP44 <jats:sub>720-1138</jats:sub> , did not. A peptide corresponding to residues 1083 to 1102, which was included in HGP44 <jats:sub>720-1138</jats:sub> but not in HGP44 <jats:sub>720-1081</jats:sub> , could bind HGP44 <jats:sub>720-1081</jats:sub> in a dose-dependent manner and effectively inhibited HGP44 <jats:sub>720-1081</jats:sub> -mediated hemagglutination, indicating that the interdomain regional amino acid sequence may function as an intramolecular suppressor of hemagglutinating activity. Analyses by solid-phase binding and chemical cross-linking suggested that HGP44 interacted with glycophorin A on the erythrocyte membrane. Glycophorin A and, more effectively, asialoglycophorin, which were added exogenously, inhibited HGP44 <jats:sub>720-1081</jats:sub> -mediated hemagglutination. Treatment of erythrocytes with RgpB proteinase resulted in degradation of glycophorin A on the membrane and a decrease in HGP44 <jats:sub>720-1081</jats:sub> -mediated hemagglutination. Surface plasmon resonance detection analysis revealed that HGP44 <jats:sub>720-1081</jats:sub> could bind to asialoglycophorin with a dissociation constant of 3.0 × 10 <jats:sup>−7</jats:sup> M. These results indicate that the target of HGP44 on the erythrocyte membrane appears to be glycophorin A. </jats:p>