The SLC36 family of proton-coupled amino acid transporters (PATs) is found within the amino acid auxin-permease (AAAP) family of the amino acid-polyamine-organocation (APC) superfamily. Members of the SLC36 family include PAT1 found to be highly expressed at the brush-border membrane of the small intestine where it is important for the absorption of dietary amino acids and PAT2 which is expressed in the renal proximal tubule and is associated with the inherited renal disorder iminoglycinuria. Functional characterisation of SLC36 family members was undertaken to investigate key differences in function and substrate specificity. Two PAT-like Drosophila transporters (CG4991 and CG7888) were also characterised functionally to expand our understanding of this family. Structural investigation of the SLC36 family was undertaken using an in silico homology modelling approach to try to determine how these key functional differences are determined at a molecular level. Until recently, insights into how such mammalian secondary active transporters function at a molecular level have been limited. However, a number of prokaryotic transporter structures within the APC superfamily have now been resolved. Members of the SLC36 family were predicted to have high structural homology to transporters with the LeuT-fold (>88%) despite limited primary sequence identity (<20%). Using the LeuT crystal structure as a template, homology models of SLC36 transporters were built. Amino acid residues of putative functional importance were identified using the position of key residues in LeuT as a guide. Site-directed mutagenesis of these residues was performed and effects on transporter function were measured by uptake of radiolabelled substrates in Xenopus laevis oocytes. This approach successfully identified functionally important residues in PAT1 and PAT2 involved in substrate occlusion (Y157 and F266 in PAT1, Y163 and F272 in PAT2), substrate binding (F159 in PAT2) and a highly conserved GXG motif critical for transport function (G61, T62 and G63 in PAT1, G67, T68 and G69 in PAT2).