Histidyl residues in peptide transporters PepT1 and PepT2 are believed to participate in proton and substrate binding and to be crucial to the transporters' functional activities. In the present study, we performed mutagenesis of rabbit PepT1. We mutated three histidine residues (H57, H111, and H121) predicted to reside in transmembrane segments, as well as tyrosine residues adjacent to H57. Functional analysis of wild-type and mutant PepT1 expressed in Xenopus oocytes, using both the radiotracer methods and two-microelectrode voltage-clamping, revealed that not only the H57 but also the aromatic residues near H57 were essential for the normal function of PepT1, in agreement with the concept that aromatic residues stabilize the charge on H+ when interacting with H57. While mutagenesis at H111 did not significantly affect the activity of PepT1, mutagenesis at H121 had profound implications. The substrate affinities for H121 mutants were decreased depending both on the charge of the substrate and the charge on the substituted residues at position 121. We propose that H57 and H121 are intimately involved in the binding of the coupling ion H+ and the recognition of transportable peptide substrates, respectively.