Amino acid ester prodrugs of antiviral and anticancer nucleoside drugs were developed to improve oral bioavailability or to reduce systemic toxicity. We studied the interaction of human concentrative nucleoside transporter (hCNT2) cloned from intestine with various amino acid ester prodrugs of floxuridine (FUdR) and 5,6-dichloro-2-bromo-1-β- D -ribofuranosylbenzimidazole (BDCRB). Na⁺-dependent uptakes of [³H]-inosine and [³H]-adenosine were measured in U251 cells transiently expressing intestinal hCNT2. FUdR significantly inhibited the uptake of both [³H]-inosine and [³H]-adenosine (60—70% of control), while its amino acid ester prodrugs including Val, Phe, Pro, Asp, and Lys esters exhibited markedly decreased inhibition potency (10—30% of control). On the other hand, BDCRB and its amino acid prodrugs markedly inhibited the uptake of both [³H]-inosine and [³H]-adenosine. Val, Phe, and Pro ester prodrugs of BDCRB showed similar inhibition capacities as parent compound BDCRB (80—90% for adenosine and 60—80% for inosine). The amino acid site of attachment (3′- and 5′-monoesters) and stereochemistry ( L - and D -amino acid esters), did not significantly affect the uptake of [³H]-inosine and [³H]-adenosine. These results demonstrate that the hCNT2 favorably interacts with BDCRB and its amino acid prodrugs, compared to those of FUdR, and that neutral amino acid esters of BDCRB have a high affinity toward this transporter. Therefore, the intestinal hCNT2 may be a target transporter as a factor for modulating oral pharmacokinetics of BDCRB prodrugs.