Fusobacterium nucleatum produces a large amount of the toxic metabolite hydrogen sulfide in the oral cavity. Here, we report the molecular basis of F. nucleatum H(2)S production, which is associated with two different enzymes: the previously reported Cdl (Fn1220) and the newly identified Lcd (Fn0625). SDS-PAGE analysis with activity staining revealed that crude enzyme extracts from F. nucleatum ATCC 25586 contained three major H(2)S-producing proteins. Two of the proteins with low molecular masses migrated similarly to purified Fn0625 and Fn1220. Their kinetic values suggested that Fn0625 had a lower enzymic capacity to produce H(2)S from L-cysteine (approximately 30%) than Fn1220. The Fn0625 protein degraded a variety of substrates containing betaC-S linkages to produce ammonia, pyruvate and sulfur-containing products. Unlike Fn0625, Fn1220 produced neither pyruvate nor ammonia from L-cysteine. Reversed-phase HPLC separation and mass spectrometry showed that incubation of L-cysteine with Fn1220 produced H(2)S and an uncommon amino acid, lanthionine, which is a natural constituent of the peptidoglycans of F. nucleatum ATCC 25586. In contrast, most of the sulfur-containing substrates tested, except L-cysteine, were not used by Fn1220. Real-time PCR analysis demonstrated that the fn1220 gene showed several-fold higher expression than fn0625 and housekeeping genes in exponential-phase cultures of F. nucleatum. Thus, we conclude that Fn0625 and Fn1220 produce H(2)S in distinct manners: Fn0625 carries out beta-elimination of L-cysteine to produce H(2)S, pyruvate and ammonia, whereas Fn1220 catalyses the beta-replacement of L-cysteine to produce H(2)S and lanthionine, the latter of which may be used for peptidoglycan formation in F. nucleatum.