In this study we describe for the first time the identification of a renal cell line that expresses the kidney-specific high-affinity H+/peptide cotransport system. The kidney cell line SKPT-0193 C1.2 was obtained by SV40 transformation of rat proximal tubular cells. The transport of the dipeptide glycylsarcosine (Gly-Sar) was studied in this cell line grown as a confluent monolayer on impermeable plastic supports. Uptake of the dipeptide was rapid and was stimulated sixfold by an inwardly directed H+ gradient, with optimal uptake occurring at an extracellular pH of 6.0. The uptake was markedly reduced by the protonophore carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone whether measured at pH 7.5 or 6.0. Intracellular acidification of the cells by NH4Cl prepulse also reduced the uptake of glycylsarcosine. The dipeptide uptake was found to be mediated by a high-affinity transport system with a Michaelis-Menten constant (Kt) of 67 +/- 2 microM and a maximal transport velocity of 1.20 +/- 0.02 nmol.10 min-1.mg protein-1. Studied over a concentration range of 5 microM to 5 mM, there was no evidence for a second saturable transport component. Di- and tripeptides, but not glycine, were strong inhibitors of glycylsarcosine uptake, indicating that these peptides also interact with the transport system with high affinity. Northern blot analysis of poly(A)+RNA from these cells using cDNA probes specific for the human intestinal peptide transporter (PEPT 1) or the human kidney-specific peptide transporter (PEPT 2) revealed that the transport system expressed in these cells is PEPT 2. It is concluded that the SKPT-0193 C1.2 cell line constitutively expresses the kidney-specific high-affinity H+/peptide cotransporter described in the proximal tubular epithelial cells of the normal kidney.