The stereoselective transport of methotrexate ( L -amethopterin, L -MTX) and its antipode ( D -amethopterin, D -MTX) by human reduced folate carrier (hRFC) has been examined in HEK293 cells expressing H27-hRFC and R27-hRFC. The uptake of both L -MTX and D -MTX increased as the extracellular pH increased from 6.0 to 7.4. The initial uptake rate of L -MTX into the H27- and R27-hRFCs of the HEK293 cells followed Michaelis–Menten kinetics with K _m values of approximately 0.24 and 0.47 µ M , respectively. Dixon plots revealed that the [³H]- L -MTX uptake mediated by the H27- and R27-hRFCs was inhibited competitively by unlabeled L -MTX with K _i values of approximately 0.1 and 0.5 µ M , respectively. D -MTX also competitively inhibited the H27- and R27-hRFC mediated uptake of [³H]- L -MTX with K _i values of approximately 3.4 and 3.2 µ M , respectively. The RFC-mediated uptake clearance of L -MTX was approximately 15-fold greater than that of D -MTX in the H27-hRFC-HEK293 cells, and was more than 30-fold greater than that of D -MTX in the R27-hRFC-HEK293 cells. The results of the current study have revealed that the enantiomers of MTX enantiomers can be transported in a stereoselective manner by the H27- and R27-hRFCs because of significant differences in the affinities of the enantiomers to the hRFC.