We studied the biosynthesis of monobactams with respect to sulfur metabolism in Chromobacterium violaceum, Acetobacter sp., and Agrobacterium radiobacter. All three organisms used inorganic sulfur for monobactam production. When sulfur-containing amino acids were assayed as a source of sulfur for monobactam production, C. violaceum used cystine but not cysteine or methionine, Acetobacter sp. used all three compounds, and A. radiobacter used none. 35S from cysteine, methionine, and sodium sulfate was incorporated into monobactam by Acetobacter sp. Cell-free extracts of all three organisms were shown to possess cysteine desulfhydrase activity. In Acetobacter sp., this activity was constitutive, required pyridoxal phosphate, and had a pH optimum of 9.5. Extensive loss of 3H from L-[3-3H]cysteine was seen upon desulfhydration; no evidence of serine formation was found. Active sulfate was formed in cell-free extracts of A. radiobacter, and, since inorganic sulfur was used by all three organisms, it is likely that the sulfamate group of monobactams is produced via active sulfate.