The crystal structure of nitrous oxide reductase, the enzyme catalyzing the final step of bacterial denitrification in which nitrous oxide is reduced to dinitrogen, exhibits a novel catalytic site, called CuZ. This comprises a cluster of four copper ions bound by seven histidines and three other ligands modeled in the X-ray structure as OH^- or H2O. However, elemental analyses and resonance Raman spectroscopy of isotopically labeled enzyme conclusively demonstrate that CuZ has one acid-labile sulfur ligand. Thus, nitrous oxide reductase contains the first reported biological copper-sulfide cluster.