Objectives: The mammalian amino acid transporter 1 (PAT1, SLC36A1), mainly expressed at the apical membrane of intestinal epithelial cells, is driven by the inwardly directed proton gradient. Little is known about structural features of the protein. The aim of this study was to determine the role of the three putative extracellular cysteine residues on transport function and their possible involvement in the formation of a disulfide bridge. Methods: Using site directed mutagenesis we exchanged the three putative extracellular cysteine residues C180, C329 and C473 individually or in combination to either alanine or serine residues. The transport function of the resulting mutants was analyzed after heterologous expression in human retinal pigment epithelial (HRPE) cells and in Xenopus laevis oocytes. Immunofluorescence studies were performed to investigate the localization of the mutant transporters within the cell. Biochemical approaches like biotinylation of free sulfhydryl groups using maleimide-PEG₁₁-biotin and protein mobility assays on SDS-PAGE under reducing and nonreducing conditions were performed to prove the existence of a disulfide bridge within hPAT1. Results: Treatment with the reducing reagent dithiothreitol impaired transport function of hPAT1 wildtype protein. Replacement of the two highly conserved cysteine residues C180 and C329 abolished the transport function of hPAT1. Substitution of the third putative extracellular non-conserved cysteine residue C473 did not affect transport function. All mutants were expressed at the plasma membrane. Conclusions: The inhibitory effect of DTT on transport function implies that a disulfide bond is formed and can be reduced. Biotinylation of free sulfhydryl groups and SDS-PAGE analysis of transporter mutants provided direct evidence for the existence of an essential disulfide bond between C180 and C329. This disulfide bridge might be involved in forming or stabilizing the substrate binding site.