The stereoselective transport of methotrexate (L-amethopterin, L-MTX) and its enantiomer (D-amethopterin, D-MTX) by the rat proton-coupled folate transporter (rPCFT) were examined using rPCFT-expressing HEK293 cells. The initial rate of uptake of [3H]-L-MTX by the rPCFT followed Michaelis-Menten kinetics, with a Km value of 2.1 µM. Dixon plots revealed that the uptake of L-MTX by the rPCFT was inhibited in a competitive manner by unlabeled L-MTX and D-MTX, with Ki values of approximately 1.3 and 150 µM, respectively. The initial rate of uptake of D-MTX by the rPCFT also followed Michaelis-Menten kinetics, with a Km value of 190 µM. The results of the current study demonstrate that the different enantiomers of MTX are transported in a highly stereoselective manner by the rPCFT, with the uptake clearance of L-MTX being approximately 46-fold greater than that of D-MTX. The observed stereoselectivity of the rPCFT was found to be comparable with that of the human PCFT.