Objective: The proton-coupled amino acid transporter 2 (PAT2) is a Na⁺ independent amino acid/H⁺-symporter. Prototypical substrates are small neutral amino acids such as L-alanine, L-proline and glycine. PAT2 was first cloned from a murine cDNA library (Boll et al. 2002). Previous studies were carried out mainly using the rat and murine PAT2. hPAT2 was localized to the apical membrane of S1 segments of human proximal tubule cells (Bröer et al. 2008). So far there were no reports on mechanism and specificity of the human PAT2. Methods and Results: We cloned hPAT2 from a testis cDNA library (Boll et al. 2003) in the pcDNA3 expression vector and expressed hPAT2 transiently in HeLa-cells. 24 h after transfection transport of L-[³H]proline was studied. L-[³H]Proline uptake was linear for 2 min, stimulated by an inwardly directed H⁺ gradient and saturable with a Michaelis constant of 0.14 ± 0.01 mM. We then tested several known and new potential substrates for their ability to inhibit L-[³H]proline uptake. For effective inhibitors, inhibition constants (K_i) were determined. To study whether or not these compounds are transported themselves by hPAT2, we injected hPAT2-cRNA in X. laevis oocytes. After two days, two-electrode voltage clamp experiments were performed to record inwardly directed currents evoked by addition of substrates. Conclusion: Our results show that the mechanism and substrate specificity of human PAT2 is similar to that from mouse and rat. There are, however, also decisive differences. We also identified new PAT2 substrates and we found differences between the substrate specificity of human PAT2 and human PAT1. Boll, M., Foltz, M., Rubio-Aliaga, I., et al. 2002. J Biol Chem 277, 22966-22973 Boll, M., Foltz, M., Rubio-Aliaga, I. & Daniel, H. 2003. Genomics 82, 47-56 Bröer, S., Bailey, C.G., Kowalczuk, S., et al. 2008. J Clin Invest 118, 3881-3892 begin_of_the_skype_highlighting