The proton-coupled amino acid transporters PAT1 (slc36a1) and PAT2 (slc36a2) are members of the SLC36 family of mammalian solute carriers (Thwaites & Anderson, 2011). PAT1 is expressed at the brush-border membrane of the small intestine whereas PAT2 is found at the luminal surface of the proximal convoluted tubule (Anderson et al. 2004, Bröer et al. 2008). Although both carriers transport a broad range of dipolar amino acids and derivatives, there are clear differences in substrate selectivity and affinity suggesting differences in substrate recognition and translocation. Using an in silico modelling approach, a high degree of structural homology (>97%) for PAT2 was detected against crystal structures for a number of apparently unrelated prokaryotic transporters. Homology modelling against the LeuT template (Yamashita et al. 2005) identified that the cysteine residues in PAT2 in extracellular loops 2 (C185) and 4 (C334) are likely in close spatial proximity. These cysteine residues are conserved in human, rat and mouse PAT1 and PAT2. In human PAT1, these residues form a disulphide bond that is essential for function (Dorn et al. 2009). The purpose of this investigation was to determine whether a similar disulphide bond was found in PAT2 and what role it might play in transporter function. Site-directed mutagenesis of rat PAT2 was performed and [3H]amino acid uptake measured in Xenopus laevis oocytes in the presence or absence of the reducing agent DTT. The results were compared with those in mouse PAT1 and the D. melanogaster ortholog CG1139 (which does not possess cysteine residues at equivalent positions). [3H]Proline uptake (mean ± SEM, n=17-20) in the presence of 30mM DTT showed a 90% reduction in PAT1 (3.42±0.34 pmol.oocyte-1 to 0.37±0.06 pmol.oocyte-1 ), 50% reduction in PAT2 (4.67±0.41pmol.oocyte-1 to 2.37±0.25 pmol.oocyte-1 ) but no change in CG1139 (8.42±0.99 pmol.oocyte-1 and 8.09±1.11 pmol.oocyte-1). In PAT1, replacement of either C179 or C328 with serine or alanine reduced amino acid uptake by 70-80% whereas only a 55-60% reduction was observed in the equivalent PAT2 C185 and C334 mutants. Confocal microscopy revealed no apparent differences in membrane localisation. The double PAT1 mutant C179A/C328A was inactive whereas the double PAT2 mutant C185A/C334A retained ~50% activity. The PAT2 double mutant was insensitive to 30mM DTT. These observations demonstrate that conserved extracellular cysteine residues in PAT1 and PAT2 form disulphide bonds that appear to be essential for PAT1 and important in PAT2 for optimal function. The role of the disulphide bridge is not known but is likely important in optimisation of substrate binding and translocation. The relative dependence of PAT1 and PAT2 on the disulphide bond may partly account for differences observed in transporter function.