The effects of mutations in the charge pair residues R282 and D341 of the rabbit oligopeptide transporter PepT1 have been studied using electrophysiology in mRNA-injected Xenopus oocytes. Substitution of Arg282 with Asp or Ala produced a shift towards more positive potentials in the characteristics of charge movement with respect to the wild-type form. Conversely replacement of Asp341 with Arg reduced both kind of currents and produced a negative shift of the charge movement properties. The presteady-state and transport currents of the mutants remained pH-sensitive. All three mutants were correctly localized on the cell membrane. In addition R282D and R282A were able to generate conspicuous outward currents whose reversal potential was affected by external pH and by substrate concentration, suggesting that the mutants still translocate protons and substrate as a complex. The apparent affinity for the substrate was only moderatly reduced in the functional mutants. The results support the idea that R282 and D341 play the role of electrostatic gates in the PepT1 transport cycle.