Besides its important role in folate homeostasis, membrane transport is a critical determinant of the antitumor activities of antifolate therapeutics used in cancer chemotherapy, such as methotrexate (MTX) and an exciting new generation of antifolates typified by Tomudex and Pemetrexed. Further, impaired cellular uptake of antifolates is a frequent mode of drug resistance. The ubiquitously expressed transporter termed the reduced folate carrier (RFC) is the best characterized of the folate transport systems in mammalian cells. This chapter summarizes recent significant advances involving the molecular structure of the RFC protein, including its topology and glycosylation, as well as identification of its functionally and structurally important domains and amino acids. Insights into these features have been gleaned by epitope insertion and accessibility methods, and by mutating specific amino acids, through selection of antifolate resistant cells with impaired RFC function, and by site-directed mutagenesis. The recent expression of a functional “cysteine-less” human RFC has permitted the use of scanning cysteine mutagenesis and thiol modification techniques for mapping membrane topology and surface accessibilities, and for identifying functionally important domains in the RFC protein.