The proton-coupled folate transporter (PCFT, SLC46A1) is required for intestinal folate absorption and folate homeostasis in humans. A homology model of PCFT, based upon theEscherichia coliglycerol 3-phosphate transporter structure, predicted that PCFT transmembrane domains (TMDs) 1, 2, 7, and 11 form an extracellular gate in the inward-open conformation. To assess this model, five residues (Gln(45)-TMD1, Asn(90)-TMD2, Leu(290)-TMD7, Ser(407)-TMD11 and Asn(411)-TMD11) in the predicted gate were substituted with Cys to generate single and nine double mutants. Transport function of the mutants was assayed in transient transfectants by measurement of [(3)H]substrate influx as was accessibility of the Cys residues to biotinylation. Pairs of Cys residues were assessed for spontaneous formation of a disulfide bond, induction of a disulfide bond by oxidization with dichloro(1,10-phenanthroline)copper (II) (CuPh), or the formation of a Cd(2+)complex. The data were consistent with the formation of a spontaneous disulfide bond between the N90C/S407C pair and a CuPh- and Cd(2+)-induced disulfide bond and complex, respectively, for the Q45C/L290C and L290C/N411C pairs. The decrease in activity induced by cross-linkage of the Cys residue pairs was due to a decrease in the influxVmaxconsistent with restriction in the mobility of the transporter. The presence of folate substrate decreased the CuPh-induced inhibition of transport. Hence, the data support the glycerol 3-phosphate transporter-based homology model of PCFT and the presence of an extracellular gate formed by TMDs 1, 2, 7, and 11.