The functional and structural basis of reverse operation of the intestinal oligopeptide transporter PepT1 have been studied by electrophysiological methods in Xenopus laevis oocytes expressing the wild-type and mutated forms of this protein. Using brief (< 250 ms) pulses from a negative holding potential, wild-type PepT1, and more evidently, mutants in Arg282, exhibit large outward currents in presence of the neutral substrate GlyGln. In all isoforms the reversal potential of these currents is affected by both pH and substrate concentration, confirming a coupled proton-substrate transport in the wild-type and in the mutants as well. However, long-lasting voltage-clamp and current-clamp experiments show that the outward currents are only temporary, and may reflect accumulation and/or depletion of substrate complex near the membrane. Properly modified protocols designed to avoid this phenomenon allow a more accurate calculation of the kinetic parameters of the transport. The role of Arg282 in the reverse transport was also investigated using differently charged dipeptides at pH 7.5. The positively-charged Lys-Gly dipeptide showed relatively enhanced transport currents in the R282D and R282A mutants,. On the contrary, the negative substrate Gly-Asp elicited modest currents in all isoforms. The results suggest that R282 is relevant in determining charge-based substrate selectivity.