Hydrogen sulfide (H(2)S) is a major metabolic end product detected in deep periodontal pockets that is produced by resident periodontopathic microbiota associated with the progression of periodontitis. Treponema denticola, a member of the subgingival biofilm at disease sites, produces cystalysin, an enzyme that catabolizes cysteine, releasing H(2)S. The metabolic pathway leading to H(2)S formation in periodontal pockets has not been determined. We used a variety of thiol compounds as substrates for T. denticola to produce H(2)S. Our results indicate that glutathione, a readily available thiol source in periodontal pockets, is a suitable substrate for H(2)S production by this microorganism. In addition to H(2)S, glutamate, glycine, ammonia, and pyruvate were metabolic end products of metabolism of glutathione. Cysteinyl glycine (Cys-Gly) was also catabolized by the bacteria, yielding glycine, H(2)S, ammonia, and pyruvate. However, purified cystalysin could not catalyze glutathione and Cys-Gly degradation in vitro. Moreover, the enzymatic activity(ies) in T. denticola responsible for glutathione breakdown was inactivated by trypsin or proteinase K, by heating (56 degrees C) and freezing (-20 degrees C), by sonication, and by exposure to N alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK). These treatments had no effect on degradation of cysteine by the purified enzyme. In this study we delineated an enzymatic pathway for glutathione metabolism in the oral spirochete T. denticola; our results suggest that glutathione metabolism plays a role in bacterial nutrition and potential virulence expression.