Methotrexate (MTX), the antifolate drug widely used as both an anticancer chemotherapeutic drug and as an immunosuppressive agent, mimics natural folates to inhibit critical cellular biosynthetic pathways. One of the most important determinants of cellular sensitivity to MTX is the degree to which this drug is internalized by cancer cells, and one of the major pathways of folate uptake results from the activity of the reduced folate carrier (RFC). Decreased RFC activity has been associated with several models of transport-mediated MTX resistance. Recently, the rodent and human genes which encode this protein have been isolated (RFC1), and defects in the expression of RFC1 genes have been identified in transport-deficient, MTX-resistant cell lines. Therefore, these studies have demonstrated the importance of RFC1 expression in transport-mediated antifolate drug resistance. In addition, however, studies of both MTX uptake in cancer cells and of folate transport in physiologic systems indicate that there are other proteins with uptake characteristics similar to RFC, and which maybe encoded by genes other than RFC1.