The Menkes (MNK) protein is a vital component of copper homeostasis in mammalian cells. In this paper we provide the first biochemical evidence that the MNK protein functions as a copper-translocating P-type ATPase in mammalian cells. The enzyme activity in membrane vesicles prepared from Chinese hamster ovary cells overexpressing MNK was ATP-dependent, correlated with the amount of MNK and followed Michaelis-Menten kinetics with respect to copper. The copper transport was observed only under reducing conditions suggesting MNK transports Cu(I). This study opens the way to detailed structure-function studies and assessment of functional MNK derived from patients with Menkes disease.