The human proton/oligopeptide cotransporters, hPEPT1 and hPEPT2, have been targeted to enhance the bioavailability of drugs and prodrugs. Previously, we established the mechanisms of drug transport by hPEPT1. Here, we extend these studies to hPEPT2. Major variants hPEPT2*1 and hPEPT2*2 were expressed in Xenopus oocytes, and each was examined using radiotracer uptake and electrophysiological methods. Gly-Sar, the -lactam antibiotics ampicillin, amoxicillin, cephalexin and cefadroxil, and the antineoplastics -aminolevulinic acid (-ALA) and bestatin induced inward currents, indicating that they are transported. Variations in transport rate were due to differences in affinity and in turnover rate: for example, cefadroxil was transported with higher apparent affinity but at a lower maximum velocity than Gly-Sar. Transport rates were highest at pH 5, and decreased significantly as the external pH was increased. Our results strongly suggest that the protein does not operate as a cotransporter in tissues where there is little or no pH gradient, such as choroid plexus, lung or mammary gland. In the absence of substrates rapid voltage jumps produced hPEPT2 capacitive currents at pH 7. These transients were significantly reduced at pH 5 but recovered upon addition of substrates. The 7-state, ordered kinetic model previously proposed for hPEPT1 accounts for the steady-state kinetics of neutral drug and dipeptide transport by hPEPT2. The model also explains the capacitive transients, the striking difference in presteady-state behavior between hPEPT2 and hPEPT1, and differences in turnover numbers for Gly-Sar and cefadroxil. No functional differences were found between the common variants hPEPT2*1 and hPEPT2*2.