The present study examined the possible role of transporters in the drug-drug interactions between methotrexate (MTX) and nonsteroidal anti-inflammatory drugs (NSAIDs) in the renal uptake process of MTX. MTX is recognized by reduced folate carrier (RFC-1) and rat organic anion transporters (rOat1 and rOat3) as a substrate. Uptake of MTX by kidney slices was saturable and inhibited potently by dibromosulfophthalein. Folate and benzylpenicillin (PCG) inhibited the uptake by 30 to 40% and 40 to 50% of the total saturable uptake of MTX by kidney slices, respectively, whereas the effect of p-aminohippurate (PAH) was minimal at the concentration selective for rOat1. In contrast, the uptake of 5-methyltetrahydrofolate by the kidney slices was inhibited by MTX, folate, and dibromosulfophthalein, but not by PAH and PCG. These results suggest that rOat3 and RFC-1 are almost equally involved in the uptake of MTX by the kidney slices, whereas RFC-1 is responsible for the renal uptake of 5-methyltetrahydrofolate. NSAIDs, except salicylate, were potent inhibitors of rOat3 (K i of 1.3-19 M), but weak inhibitors of RFC-1 (K i of 70 -310 M). This is in a good agreement with the biphasic inhibition profiles of NSAIDs for the uptake of MTX by kidney slices. These results suggest that the renal uptake of MTX is not so greatly affected by NSAIDs as expected from the inhibition of rOat3-mediated transport.Drug-drug interactions involving metabolism and/or excretion process prolong the plasma elimination half-lives, leading to the accumulation of drug in the body after repeated administration, and potentiate pharmacological/adverse effects. Investigating the mechanism of a drug-drug interaction is important as far as achieving safer chemotherapy is concerned. Transporters are one of the determinant factors governing drug disposition together with metabolic enzymes and have been regarded as important drug-drug interaction sites in the body (Ito et al., 1998). Recent progress in molecular biological research has shown that many types of transporters play important roles in the tissue uptake and/or subsequent secretion of drugs in the liver and kidney, and enables us to examine the incidence of drug-drug interactions involving membrane transport based on the contribution of the transporters to the membrane transport of the drugs in question.Methotrexate (MTX) is an analog of natural folate and has been used not only for the treatment of neoplastic diseases (Evans et al., 1986) but also autoimmune diseases including rheumatoid arthritis (Giannini et al., 1992) and psoriasis (Chladek et al., 1998). Numerous case reports have indicated that coadministration of MTX with penicillin, probenecid, and nonsteroidal anti-inflammatory drugs (NSAIDs) cause drug-drug interactions, which affect the pharmacokinetic parameters of MTX (Furst, 1995). To date, several potential sites have been proposed for these drug-drug interactions: an increase in the unbound fraction of MTX (Tracy et al., 1992), a decrease in the urine flow rate resulting from ...