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The hPEPT1 cDNA cloned from human intestine (Liang, R., Fei, Y.-J., Prasad, P. D., Ramamoorthy, S., Han, H., Yang-Feng, T. L., Hediger, M. A., Ganapathy, V., and Leibach, F. H. (1995) J. Biol. Chem. 270, 6456-6463) encodes a H⁺/oligopeptide cotransporter. Using two-microelectrode voltage-clamp in Xenopus oocytes expressing hPEPT1, we have investigated the transport mechanisms of hPEPT1 with regard to voltage dependence, steady-state kinetics, and transient charge movements. The currents evoked by 20 mM glycyl-sarcosine (Gly-Sar) at pH 5.0 were dependent upon membrane potential (V_m) between −150 mV and +50 mV. Gly-Sar-evoked currents increased hyperbolically with increasing extracellular [H⁺], with Hill coefficient ≈1, and the apparent affinity constant (K_0.5^H) for H⁺ was in the range of 0.05-1 μM. K_0.5 for Gly-Sar (K_0.5^GS) was dependent upon V_m and pH; at −50 mV, K_0.5^H was minimal …