It has been suggested that Porphyromonas gingivalis may possess more than one hemagglutinin. We have previously reported the cloning of a gene (hagA) that encodes a hemagglutinin. In this study we report the cloning, characterization, and sequencing of a second gene (hagB) that encodes a protein that also appears to be involved in hemagglutination. Antiserum to the clone (ST 7) was found to inhibit hemagglutination by P. gingivalis 381, and hemagglutinating inhibition activity of anti-P. gingivalis antiserum was reduced by adsorption of the antiserum with cells of clone ST 7. Restriction mapping and Southern analysis indicates there is little or no DNA homology between this cloned 4.8-kb HindIII DNA fragment and a cloned hemagglutinin gene we have previously described. Minicell analysis of the cloned P. gingivalis chromosomal DNA fragment revealed that the major gene product is a 49-kDa protein. Immunoaffinity chromatography using purified rabbit immunoglobulin G against the cloned protein resulted in the purification of a major reactive 49- to 50-kDa protein from a P. gingivalis cell lysate. Nucleotide sequence analysis revealed the hagB open reading frame to be 1053 nucleotides in length with a mol% G+C of 59.9% coding for a protein of 350 residues with a calculated molecular weight of 39.375 kDa. This protein was also determined to be basic and hydrophilic and to contain a potential signal peptide. Comparison of both the nucleotide and derived amino acid sequences with computer-based databases did not reveal any significant homologies between habB and any other previously sequenced genes.