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 (Vm) between -150 mV and +50 mV. Gly-Sar-evoked currents increased hyperbolically with increasing extracellular [H+], with Hill coefficient approximately 1, and the apparent affinity constant (K0.5H) for H+ was in the range of 0.05 1 microM. K0.5 for Gly-Sar (K0.5GS) was dependent upon Vm and pH; at -50 mV, K0.5H was minimal (approximately 0.7 mM) at pH 6.0. Following step-changes in Vm, in the absence of Gly-Sar, hPEPT1 exhibited H+-dependent transient currents with characteristics similar to those of Na+-coupled transporters. These charge movements (which relaxed with time constants of 2-10 ms) were fitted to Boltzmann relations with maximal charge (Qmax) of up to 12 nC; the apparent valence was determined to be approximately 1. Qmax is an index of the level of transporter expression which for hPEPT1 was in the order of 1011/oocyte. In general our data are consistent with an ordered, simultaneous transport model for hPEPT1 in which H+ binds first.