Folate cofactors are one-carbon donors essential for the biosynthesis of purines and thymidylate. Mammalian cells are devoid of folate biosynthesis and are therefore folate auxotrophs that take up folate vitamins primarily via the reduced folate carrier (RFC). In this study, we showed that the human RFC (hRFC) gene can serve as a novel selectable marker for the overproduction of recombinant proteins. Toward this end, a hemagglutinin (HA) epitope tagged hRFC (hRFC-HA) was introduced into a bicistronic vector (pIRES2-EGFP), upstream of an enhanced green fluorescent protein (EGFP) reporter gene. Chinese hamster ovary cells deficient in RFC activity were isolated and transfected with this construct, followed by gradual deprivation of leucovorin, the sole folate source in the growth medium. Only cells with hRFC-HA overexpression were able to take up leucovorin and thereby survive these selective conditions. Western blot and immunofluorescence analyses confirmed that the hRFC-HA was overexpressed at extremely high levels, properly glycosylated and sorted out to the plasma membrane. This resulted in a approximately 450-fold increase in [3H]methotrexate influx and approximately 100-fold increased sensitivity to methotrexate, relative to untransfected RFC-deficient cells. Flow cytometric analysis consistently revealed that EGFP was overexpressed approximately 100-fold above the autofluorescence level. Overproduction of hRFC-HA and EGFP was stably maintained for at least 2 months in a constant concentration of leucovorin. These results establish a novel RFC-based metabolic selection system for the efficient overexpression of recombinant proteins. Furthermore, the possible implications to subcellular transporter localization and restoration of MTX sensitivity in drug-resistant tumors by RFC-based gene therapy are discussed.