Bacteroides forsythus is a fastidious anaerobic gram-negative organism associated with various forms of periodontal disease. It is dependent on N-acetylmuramic acid for growth. A method for rapid presumptive identification of human-derived strains of B. forsythus is presented, based on the following eight criteria: (i) positive activity for alpha-glucosidase, (ii) positive activity for beta-glucosidase, (iii) positive activity for sialidase, (iv) positive activity for trypsinlike enzyme, (v) negative indole production, (vi) requirement for N-acetylmuramic acid, (vii) colonial morphology, and (viii) gram stain morphology from blood agar medium deficient in N-acetylmuramic acid. Enzymes were assayed with rapid filter paper spot tests based on fluorogenic substrates (4-methylumbelliferone derivatives and N alpha-carbobenzoxy-L-arginine-7-amino-4-methylcoumarin hydrochloride). Gas-liquid chromatography analysis of the metabolic products of B. forsythus grown in peptone yeast extract broth supplemented with N-acetylmuramic acid and heat-inactivated horse serum revealed predominant amounts of acetate, propionate, butyrate, isovalerate, and phenyl acetate, with minor amounts of isobutyrate and succinate. The described presumptive identification scheme facilitated recognition of four strains of B. forsythus which were isolated from subgingival plaque samples from monkeys (Macaca fascicularis). With the exception of indole production, these organisms were essentially identical to the human strains of B. forsythus for all phenotypic and genotypic characteristics examined.