We constructed engineered CHO strains that can be used for high-level production of foreign proteins by gene-targeting. After transfecting dihydroforate reductase (DHFR)-deficient CHO cells with a plasmid carrying a loxP-green fluorescent protein (GFP) fusion gene and a DHFR gene, we screened colonies by fluorescent intensity. We selected 16 clones that expressed high levels of GFP and carried one copy of the plasmid in their chromosomes and treated them with methotrexate (MTX) to examine their ability for DHFR-mediated gene amplification. Two clones, MK1 and MK2, showed increased GFP expression upon gene amplification. In those clones, the loxP-GFP gene was integrated at a transcription-active, DHFR-mediated, gene-amplifiable locus in the chromosomes. A gene-targeting vector, carrying a loxP-fused hygromycin-resistance gene, was constructed to target desired genes in chromosomal loxP by Cre recombinase-mediated site-specific recombination. Using this cell-vector system, we could reproducibly obtain high producers of recombinant proteins by gene-targeting and gene amplification. In human monoclonal antibody production, after gene-targeting of loxP in MK2 and gene amplification with MTX, the MTX-resistant colonies showed high levels of antibody production. The most productive clone was able to produce 160 mg/l in 7 days in a low-protein medium in a spinner-flask.