Three copper-resistant variants of cultured Chinese hamster ovary (CHO) cells were isolated and each was shown to accumulate less intracellular copper than the parental cells when grown in copper-supplemented media. The reduced copper accumulation was related to enhanced copper efflux. As cultured cells from patients with Menkes disease (mutations in MNK; ATP7A gene) accumulate copper, probably due to defective copper efflux, we investigated the possible role of the MNK gene in the molecular basis of copper resistance. We found increased MNK mRNA and MNK protein in all three resistant variants. The MNK protein, which has not been previously demonstrated experimentally in mammalian cells, was observed to have an apparent molecular weight of 178 kDa on SDS gels. The degree of increase in MNK mRNA and protein correlated well with the level of copper resistance and extent of copper efflux. By Southern blot and FISH analysis we determined that the molecular basis for overexpression of MNK was genomic amplification of the MNK gene. These data, combined with the clinical and cellular phenotype in Menkes disease, provide strong evidence that the MNK protein is involved in transmembrane copper efflux, and demonstrate a new system of gene amplification in mammalian cells.