Amino acids are essential for normal biological function. The efficient supply of amino acids is mediated by specialised membrane transport proteins. A proton electrochemical gradient provides the driving force for amino acid transport via the proton-coupled amino acid transporters PAT1 and PAT2. The objective of this study was to further our understanding of the function of PAT1 and PAT2 at the molecular and cellular level. When heterologously expressed in isolation using Xenopus laevis oocytes, radiolabelled amino acid uptakes revealed that mouse PAT1, rat PAT2 and the Drosophila CG1139 protein functioned as pH-dependent (proton-coupled), sodium-independent transporters. CG1139 closely resembled PAT1 in terms of its pH-dependence, substrate affinity (mM range) and specificity, with a notable exception being that L-serine (a relatively poor PAT1/PAT2 substrate) was an excellent CG1139 substrate. PAT1 and TauT mediate the uptake of the conditionally-essential amino acid taurine across the apical membrane of human intestinal (Caco-2) epithelial cell monolayers. Exposure of Caco-2 cell monolayers to high, dietary, concentrations of taurine reduced the transport activity of TauT but PAT1-mediated taurine uptake was maintained or increased. This may reflect a physiological adaptive response designed to optimise taurine absorption from the small intestine following ingestion of a protein-rich meal. The endogenous expression of multiple amino acid transporters with overlapping substrate specificity can constrain the study of individual transport proteins in intact tissues. Using a range of complementary techniques, 5-hydroxy-L-tryptophan and α-methyl-D,L-tryptophan were identified as novel non-transported inhibitors of PAT2. α-Methyl-D,L-tryptophan failed to inhibit PAT1 and may prove useful as an experimental tool for future studies designed to discriminate between PAT1 and PAT2 transport activity in vivo. Homology modelling of the slc36 transporters identified PAT2 residues putatively involved in: substrate binding (G67, T68, G69, E275); substrate occlusion (Y163 and F272); and stabilisation of the substrate binding site (C185 and C334). Substitution with homologous CG1139 residues conferred on the PAT2 mutants G64A and F159I the ability to transport the CG1139 substrate L-serine. Homology modelling may provide insights as to the structural defects in related SLC transporters implicated in disorders of amino acid transport.