The transport of 10 amino acid ester prodrugs of levovirin (LVV) was investigated in the human intestinal Caco-2 cell line in order to overcome the poor oral bioavailability of LVV, an investigational drug for the treatment of hepatitis C infection. The prodrugs were designed to improve the permeability of LVV across the intestinal epithelium by targeting the di/tri-peptide carrier, PepT1. Caco-2 cell monolayers were employed to study the transport and hydrolysis properties of the prodrugs. Among all mono amino acid ester prodrugs studied, the LVV-5'-(L)-valine prodrug (R1518) exhibited the maximum increase (48-fold) in permeability with nearly complete conversion to LVV within 1 h. Di-amino acid esters did not offer significant enhancement in permeability comparing with mono amino acid esters and exhibited slower conversion to LVV in Caco2 cell monolayers. Pharmacokinetic screening studies of the prodrugs in rats yielded the highest fold increase (6.9-fold) of AUC with R1518 and in general displayed a similar trend to that observed in increases of permeability in Caco-2 cells. Mechanisms involved in the Caco-2 cell transport of R1518 were also investigated. Results of bi-directional transport studies support the involvement of carrier-mediated transport mechanisms for R1518, but not for the LVV-5'-(D)-valine prodrug or LVV. Moreover, the permeability of R1518 was found to be proton dependent. PepT1-mediated transport of R1518 was supported by results of competitive transport studies of R1518 with the PepT1 substrates enalapril, Gly-Sar, valganciclovir, and cephalexin. R1518 was also found to inhibit the permeability of valganciclovir and cephalexin. These results suggest that R1518 is a PepT1 substrate as well as an inhibitor.