Folate cofactors serve as one-carbon donors in the de novo biosynthesis of purines and thymidylate (1). As such, normal and neoplastic dividing cells have an absolute folate requirement for DNA replication (1). Disruption of folate biosynthesis with folic acid antagonists (i.e. antifolates) is the pharmacological basis for the antitumor activity of methotrexate (MTX) 1 and various antifolates (2). Because mammalian cells are devoid of folate biosynthesis, they rely on folate vitamin uptake from exogenous sources. Membrane transport of folates and MTX is mediated by several systems (3, 4): (a) the reduced folate carrier (RFC) is the major uptake route that functions as a bi-directional anion exchanger (5, 6) taking up folates through an antiport exchange mechanism with intracellular organic phosphates (7); (b) folate receptors mediate the unidirectional uptake of folate cofactors into mammalian cells via an endocytotic process (8); and (c) an apparently independent transport system with optimal folate uptake activity at low pH (9 -11).Apart from RFC, efflux of folates and MTX (12, 13) is mediated by multidrug resistance proteins (MRP) MRP1-4 (14 -19), which belong to the ATP-binding cassette superfamily (20,21). Members of the MRP family, currently comprising nine genes (i.e. MRP1-9), function as ATP-driven efflux transporters of various natural product anions and acidic charged drug conjugates (14, 15). Mammalian cells transfected with MRP1-4 accumulate decreased levels of MTX and consequently display resistance to this drug, particularly upon short term drug exposure (16 -19). Membrane vesicles isolated from MRP1-and MRP2-transfected cells exhibit ATP-dependent transport of MTX (16). Detailed kinetic analysis of folic acid, leucovorin (LCV; 5-formyl-tetrahydrofolate) and MTX transport into MRP1-and MRP3-rich membrane vesicles reveals K m values in the low millimolar range (22). Hence, the free intracellular level of folates and antifolates including MTX is determined by the net activities of these influx (i.e. RFC) and efflux (RFC and MRP) transport pathways.CEM-7A is a human leukemia CCRF-CEM subline previously established by gradual deprivation of LCV from the growth medium (23), resulting in RFC gene amplification (24) and carrier overexpression (23,24). Consequently, CEM-7A cells displayed a marked increase in the influx of MTX and LCV accompanied by a comparable increase in the steady-state transmembrane gradient of MTX (23). Surprisingly, however, there was no increase in the efflux rate constant for MTX (23). This is in contrast with previous studies (25) 1 The abbreviations used are: MTX, methotrexate; MRP, multidrug resistance protein; RFC, reduced folate carrier; LCV, Leucovorin (5-formyl-tetrahydrofolate); TMQ, trimetrexate; BCRP, breast cancer resistance protein; Pgp, P-glycoprotein; NHS-MTX, N-hydroxysuccinimide ester of MTX; AM, acetoxymethyl ester; HBS, Hepes-buffered saline.