Trypsin-like protease activity, hemagglutination activity, and accumulation of heme-containing compounds (black pigment) are considered to be virulence factors of Porphyromonas gingivalis. Transposon-mutagenesis was used for the first time to isolate pigment-deficient mutants. These mutants exhibited simultaneous deficiency in trypsin-like protease activity and hemagglutination activity. Two major membrane-associated proteins, observed by SDS-PAGE with the parent strain, were essentially absent from the mutant strains. Immunoblot analysis indicated that these two proteins correspond to putative hemagglutinin and hemagglutinin/protease products of P. gingivalis. Each mutant contained only one transposon insertion, thus the pleiotropic phenotype resulted from single site-specific mutations. The results indicate that trypsin-like protease activity is required for accumulation of protoheme from hemoglobin by P. gingivalis and that genetic and/or physiological linkage exists between trypsin-like protease activity and hemagglutination activity.