Valacyclovir is a prodrug of the antiviral agent acyclovir and it does not contain a peptide bond in its structure. We studied the interaction of valacyclovir with the peptide transporters in the human intestinal cell line Caco-2 and the rat kidney proximal tubular cell line SKPT which differentially express peptide transporters PEPT1 and PEPT2. The results of the studies done with these cell lines were confirmed with the cloned peptide transporters human PEPT1 and rat PEPT2, expressed heterologously in HeLa cells. The activity of the peptide transporters was assessed by measuring the uptake of radiolabeled glycylsarcosine in the presence of a H+ gradient. Valacyclovir inhibited the uptake of glycylsarcosine with an inhibition constant (Ki) of 0.49 +/- 0.04 mM in Caco-2 cells and 0.17 +/- 0.01 mM in SKPT cells. In both cell types, the inhibition was competitive. Acyclovir, in contrast to valacyclovir, did not interact with the peptide transporters. Similar results were obtained with heterologously expressed human PEPT1 and rat PEPT2. Valacyclovir inhibited the hPEPT1-mediated glycylsarcosine transport competitively with a Ki value of 0.74 +/- 0.14 mM. The rPEPT2-mediated transport of glycylsarcosine was also inhibited by valacyclovir competitively and the Ki value for the process was 0.39 +/- 0.03 mM. Acyclovir did not interact with either of these cloned peptide transporters. We conclude that valacyclovir is a substrate for the peptide transporters PEPT1 and PEPT2 and that a peptide bond is not a prerequisite for recognition as a substrate by the peptide transporters.