The human reduced folate carrier (hRFC) mediates the membrane transport of reduced folates and classical anti-folates into mammalian cells. RFC is characterized by 12 transmembrane domains (TMDs), internally oriented N and C termini, and a large central linker connecting TMDs 1-6 and 7-12. By co-expression and N-hydroxysuccinimide methotrexate (Mtx) radioaffinity labeling of hRFC TMD 1-6 and TMD 7-12 half-molecules, combined with endoproteinase GluC digestion, a substrate binding domain was previously localized to within TMDs 8-12 (Witt, T. L., Stapels, S. E., and Matherly, L. H. (2004) J. Biol. Chem. 279, 46755-46763). In this report, this region was further refined to TMDs 11-12 by digestion with 2-nitro-5-thiocyanatobenzoic acid. A transportcompetent cysteine-less hRFC was used as a template to prepare single cysteine-replacement mutant constructs in which each residue from Glu-394 to Asp-420 of TMD 11 and Tyr-435 to His-457 of TMD 12 was replaced individually by a cysteine. The mutant constructs were transfected into hRFC-null HeLa cells. Most of the 50 single cysteine-substituted constructs were expressed at high levels on Western blots. With the exception of G401C hRFC, all mutants were active for Mtx transport. Treatment with sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES) had no effect on hRFC activity for all of the cysteine mutants within TMD 12 and for the majority of the cysteine mutants within TMD 11. However, MTSES inhibited Mtx uptake by the T404C, A407C, T408C, T412C, F416C, I417C, V418C, and S419C mutants by 25-65%. Losses of activity by MTSES treatment for T404C, A407C, T412C, and I417C hRFCs were appreciably reversed in the presence of excess leucovorin, a hRFC substrate. Our results strongly suggest that residues within TMD 11 are likely critical structural and/or functional components of the putative hRFC transmembrane channel for anionic folate and anti-folate substrates.