Copper-deficient cells of Pseudomonas stutzeri strain ZoBell synthesize catalytically inactive nitrous oxide (N2O) reductase which is activated by added Cu(II) in the absence of de novo protein synthesis. The apparent Km for the activation process is 0.13 µM. Activation is temperature-dependent and is inhibited by Cd(II) (Ki 1.27 µM) and less strongly by Zn(II), Ni(II), and Co(II). The same metal ions at 20 µM have little or no effect on N2O reduction of intact cells. Apo-N2O reductase of transposon Tn5-induced nos^- mutants with defective Cu-chromophore biosynthesis is not reactivated by Cu(II). N2O reductase of Cu-sufficient and Cu-deficient wild type, and of nos^- mutants is localized in the periplasm, the latter providing the likely site of metal incorporation into the apoenzyme.