Reduced folate carrier (RFC) is the major membrane transporter for folates and antifolates in mammalian tissues. Recent studies used radioaffinity labeling with N-hydroxysuccinimide (NHS)-3H-methotrexate (MTX) to localize substrate binding to residues in transmembrane domain (TMD) 11 of human RFC. To identify the modified residue(s), seven nucleophilic residues in TMD11 were mutated to Val or Ala and mutant constructs expressed in RFC-null HeLa cells. Only Lys411Ala RFC was not inhibited by NHS-MTX. By radioaffinity labeling with NHS-3H-MTX, wild type (wt) RFC was labeled; for Lys411Ala RFC, radiolabeling was abolished. When Lys411 was replaced with Ala, Arg, Gln, Glu, Leu, and Met, only Lys411Glu RFC showed substantially decreased transport. Nine classical diamino furo[2,3-d]pyrimidine antifolates with unsubstituted α- and γ-carboxylates (1), hydrogen- or methyl-substituted α- (2, 3) or γ- (4, 5) carboxylates, or substitutions of both α- and γ-carboxylates (6, 7, 8, 9) were used to inhibit 3H-MTX transport with RFC-null K562 cells expressing wt and Lys411Ala RFCs. For wt and Lys411Ala RFCs, inhibitory potencies were in the order 4>5>1>3>2; 6-9 were poor inhibitors. Inhibitions decreased in the presence of physiologic anions. When NHS esters of 1, 2, and 4 were used to covalently modify wt RFC, inhibitory potencies were in the order 2>1>4; inhibition was abolished for Lys411Ala RFC. These results suggest that Lys411 participates in substrate binding via an ionic association with the substrate γ-carboxylate, however, this is not essential for transport. An unmodified α-carboxylate is required for high affinity substrate binding to RFC, whereas the γ-carboxyl is not essential.