1. The giant patch clamp technique in the inside-out configuration and the two-electrode voltage clamp technique were used to characterize the bidirectional transport properties of the proton-coupled peptide carrier PEPT1 expressed in Xenopus laevis oocytes. 2. The addition of the neutral dipeptide Gly-L-Gln to the cytoplasmic solution induced a net outward transport current in a membrane potential range between -80 and +60 mV, even in the absence of a pH gradient. 3. The concentration dependency of the outwardly directed transport currents followed Michaelis-Menten-type kinetics, with an apparent K0.5 of 3.28 mM (at pH 7.5 and +60 mV membrane potential). This apparent affinity is around fivefold lower than the apparent affinity measured for the inward transport mode (K0.5 of 0.70 mM (at pH 7.5 and -60 mV) under identical experimental conditions). 4. Apparent K0.5 values were strongly pH and potential dependent only on the external face for inward transport. The transport currents were potential dependent, but essentially pH independent for inward transport and only modestly altered by pH in the reverse direction. In addition to the membrane potential, the transmembrane substrate gradient acts as a driving force and contributes significantly to total transport currents. 5. The differences in apparent substrate affinity under identical experimental conditions suggest major differences in the conformation of the substrate binding pocket of PEPT1 when exposed to the external versus the internal face of the membrane. The lower affinity on the internal face allows the substrate to be released into the cytosolic compartment even in the absence of a proton-motive force. 6. Our study demonstrates for the first time that PEPT1 can transport dipeptides bidirectionally in an electrogenic and proton-coupled symport mode. When substrates are present on both sides of the membrane in sufficiently high concentrations, the direction and rate of transport are solely dependent on the membrane potential, and transport occurs symmetrically.