Mutants carrying defects in cysteine synthase A or B or both were isolated from Salmonella typhimurium LT2. Parent strains were able to grow on minimal media containing sulfate, sulfite, sulfide, or thiosulfate as sulfur sources. Mutants lacking cysteine synthase B were unable to grow on thiosulfate, whereas mutants lacking cysteine synthase A grew on the four inorganic sulfur sources described above with little difference in their growth rates. Mutants lacking both cysteine synthases failed to grow on media containing any of the inorganic sulfur sources tested. Purification of cysteine synthase B resulted in the copurification of S-sulfocysteine synthase. In addition, the two activities were also cotransduced. These activities appear to be associated with the cysM gene, and this is able to be cotransducted with the cysK gene at a high frequency. From these results, it may be concluded that thiosulfate is assimilated via S-sulfocysteine exclusively with the aid of S-sulfocysteine synthase.