AbstractStereoselectivity of the human reduced folate carrier (RFC1) was examined in Caco‐2 cells using methotrexate (l‐amethopterin or l‐MTX) and its antipode (d‐amethopterin or d‐MTX) as model substrates. The initial uptake rate of folic acid (FA) was concentration dependent, with a Km value of approximately 0.6 μM. The Eadie–Hofstee plot of the RFC1‐mediated FA uptake revealed a single component for FA uptake into Caco‐2 cells, demonstrating that only RFC1 is involved in FA uptake. l‐MTX inhibited FA uptake in a competitive manner with a Ki value of approximately 2 μM, similar to the Km value of l‐MTX. d‐MTX also competitively inhibited FA uptake with a Ki value being approximately 120 μM, indicating that the affinity of d‐MTX is ca. 60‐fold less than that of l‐MTX. The stereoselectivity of human RFC1 observed in the present study was consistent not only with the stereoselectivity of rabbit RFC1 observed in rabbit intestinal brush border membrane vesicles but also with the reported differences in oral absorption of amethopterin enantiomers in humans. © 2005 Wiley‐Liss, Inc. Chirality 17:444–449, 2005.