We cloned, by complementation of an H2S-mutant, a cluster of Salmonella typhimurium genes, phsBCDEF, that appears to be essential for the anaerobic production of hydrogen sulfide from thiosulfate. Tn5 mutagenesis and ExoIII deletion analysis showed that approx. the entire region of a 3.3-kb subclone was necessary for H2S production. Subsequent sequencing revealed the presence of five potential translationally coupled open reading frames (ORFs). Their putative protein products were confirmed by synthesis from a phage T7 expression system. Comparison of the encoded sequences with previously determined sequences suggests that these genes constitute part of a thiosulfate-reducing operon coding for a membrane-associated electron transport chain which contains proteins potentially capable of ligating iron-sulfur clusters and heme. Immediately upstream from these genes, a region encoding the C-terminal portion of an ORF (OrfA) was identified that showed a high degree of similarity to some other anaerobic terminal reductases, polysulfide reductase (PsrA) of Wolinella succinogenes and dimethylsulfoxide reductase (DmsA), formate dehydrogenase (formate-hydrogene-lyase linked) (FdhF) and nitrate reductase (NarG) of Escherichia coli.