The antifolates methotrexate and trimetrexate are potent inhibitors of the dihydrofolate reductase, an enzyme that delivers by the metabolism of folate and its reduced forms the C1-groups for the synthesis of nucleotides and amino acids. Methotrexate, which has a comparatively moderate toxicity is one of the most important clinically used chemotherapeutics for the treatment of leukemias, such as acute lymphoblastic leukemia of children. It is also used in breast cancer, squamous tumors of the head and neck, lymphomas, and choriocarcinomas. 1,2 Uptake studies of methotrexate and the natural folic acid derivatives in different tumor and normal cells have shown that at least two different carrier systems mediate the uptake of these compounds. The 40 kd folate binding protein has a high affinity for folic acid but a low affinity for reduced folates. [3][4][5][6][7][8][9][10] The corresponding complementary DNAs (cDNAs) as well as genomic clones have been isolated from various sources, 11-19 but a detailed characterization of the transporter function is still missing.Different cDNAs and genomic clones for the reduced folate carrier, which has a high affinity for the reduced folates 5-methyltetrahydrofolate, 5-formyltetrahydrofolate, and methotrexate, but a low affinity for folate [20][21][22][23][24] have been also isolated from different immortal cell lines 25-31 but the clones were not characterized in detail.The expression and the abundance of the two carriers varied in different tumor and normal cells. From uptake studies it became evident that, first, in some cell systems both transport systems work in tandem 32 whereas two independently operating carrier systems were also described. 33,34 Second, tumor cells differ from normal cells in their transport properties. [35][36][37][38][39] Results from Chello et al. 40 showed, for example, a 30-fold higher affinity for methotrexate in the mouse leukemia cell line L1210 than in freshly isolated murine intestinal cells.Hepatocytes are exceptional with respect to folate transport. In contrast to most other cells, hepatocytes express a separate carrier system for 5-methyltetrahydrofolate, which is different from the reduced folate transporter. 41,42 We recently characterized the uptake of methotrexate into freshly isolated hepatocytes 43 and showed that this chemotherapeutic is taken up by a sodium-dependent active transport system, which is inhibited by the reduced folates, dihydrofolate, and tetrahydrofolate but not by folate itself. Moreover, substrates of two well-known organic anion transport systems in the liver, the Ntcp1 44,45 and the oatp1, 46 like the bile acids taurocholate and cholate as well as xenobiotics such as bromosulfophthalein, the loop diuretic bumetanide, and the mycotoxin ochratoxin A are potent inhibitors of the hepatocellular methotrexate carrier. Because some substrates were Abbreviations: cDNA, complementary DNA; RL-Mtx, rat liver methotrexate transporter; RACE-PCR, rapid amplification of cDNA ends polymerase chain reaction; DMEM, Dulbecco modi...