We examined the peptide transport activity in renal basolateral membranes. [¹⁴C]glycylsarcosine (Gly-Sar) uptake in rat renal cortical slices was saturable and inhibited by excess dipeptide and aminocephalosporin cefadroxil. When several renal cell lines were screened for the basolateral peptide transport activity, Madin-Darby canine kidney (MDCK) cells were demonstrated to have the greatest transport activity. [¹⁴C]Gly-Sar uptake across the basolateral membranes of MDCK cells was inhibited by di- and tripeptide and decreased with decreases in extracellular pH from 7.4 to 5.0. The Michaelis-Menten constant value of [¹⁴C]Gly-Sar uptake across the basolateral membranes of MDCK cells was 71 µM. The basolateral peptide transporter in MDCK cells showed several different [¹⁴C]Gly-Sar transport characteristics in growth dependence, pH profile, substrate affinity, and sensitivities to chemical modifiers from those of the apical H⁺-peptide cotransporter of MDCK cells. The findings of the present investigation indicated that the peptide transporter was expressed in the renal basolateral membranes. In addition, from the functional characteristics, the renal basolateral peptide transporter was suggested to be distinguishable from known peptide transporters, i.e., H⁺-peptide cotransporters (PEPT1 and PEPT2) and the intestinal basolateral peptide transporter.