Black-pigmented Bacteroides (BPB) and spirochetes are associated with some forms of periodontal diseases. The enzymes produced by these bacteria may participate in the destruction of gingival and periodontal tissues. Certain proteases and peptidases are unique to Bacteroides gingivalis and Treponema denticola. Our purpose was to study the peptidases of periodontopathogens and to evaluate the use of unique peptidases for detection and identification of these bacteria. Bacteria used were BPB, Treponema, Fusobacterium, Capnocytophaga, Actinobacillus (Haemophilus), and Eikenella species. Twenty-five substrates, including mono-, di-, and tri-peptides of β-naphthylamide (β-NA) were employed for examination of peptidase activity. Clinically isolated BPB were obtained from 16 adult periodontitis patients. One hundred and ninety-three BPB strains were identified by conventional identification methods, and the peptidase activity was determined with N-Carbobenzoxy-glycyl-glycyl-L-arginine-β-naphthylamide (N-CBz-Gly-Gly-Arg-β-NA) used as a substrate. Among tested periodontopathic bacteria, only B. gingivalis and T. denticola could strongly hydrolyze some substrates such as N-CBz-Gly-Gly-Arg-β-NA and N-Benzoyl-L-valyl-glycyl-L-arginine-4-methoxy-(3-naphthylamide (Bz-Val-Gly-Arg-β-NA). In subgingival plaque samples, all patients showed BPB, and eight out of 16 patients possessed B. gingivalis by culture. One hundred and ten strains out of 193 BPB isolated were identified as B. gingivalis. Ninety-nine percent of these B. gingivalis strains identified showed N-CBz-Gly-Gly-Arg-β-NAhydrolyzing activity on a newly developed colorimetric plate assay. However, none of the other strains showed this activity in cultures of subgingival plaque which did not allow growth of spirochetes. Enzymes, such as N-CBz-Gly-Gly-Arg-peptidase and Bz-Val-Gly-Arg-peptidase, specific for B. gingivalis and T. denticola seem to be useful for rapid detection and identification of these bacteria.