The putative multi-copper oxidase CueO had previously been implicated in intrinsic copper resistance in Escherichia coli. In this report we showed that the presence of CueO in the periplasm protected alkaline phosphatase from copper-induced damage. CueO contained four. copper atoms per molecule and displayed spectroscopic properties typical of blue copper oxidases. CueO catalyzed the oxidation of p-phenylene-diamine (pPD), 2,6-dimethoxyphenol (DMP) and exhibited ferroxidase activity in vitro.