We examined the pharmacological role of the renal organic anion transporter OAT-K1, which localizes predominantly in the brush-border membranes of proximal straight tubules, in the urinary excretion of methotrexate and the possibility of its contribution to "folinic acid rescue." With Madin-Darby canine kidney (MDCK) cells stably transfected with OAT-K1 cDNA, OAT-K1-mediated methotrexate accumulation was inhibited in the presence of various folic acid derivatives. These derivatives included aminopterin, 5-methyltetrahydrofolic acid, unlabeled methotrexate, folinic acid (citrovorum factor, leucovorin), and folic acid with apparent inhibition constant values of 0.5, 1.2, 1.8, 8.2, and 14.1 microM, respectively. In contrast, 10 microM taurocholic acid and sulfobromophthalein did not inhibit OAT-K1-mediated methotrexate accumulation. In addition, methotrexate efflux was stimulated in the presence of inwardly directed gradients of aminopterin, 5-methyltetrahydrofolic acid, unlabeled methotrexate, folinic acid, and folic acid, but not of uric acid, taurocholic acid, and glutathione, indicating that OAT-K1-mediated methotrexate efflux is stimulated by a folic acid derivatives exchange. In conclusion, OAT-K1 was suggested to enhance the apical efflux of highly accumulated methotrexate in tubular epithelial cells and contribute at least in part to folinic acid rescue by exchanging intracellular methotrexate for extracellular folinic acid.