Various antimetabolites of nucleobase analogues, such as 6-mercaptopurine (6-MP), 6-thioguanine (6-TG) and 5-fluorouracil (5-FU), are used for cancer treatments. The first step in nucleobase analogue drug therapy is entry of these compounds into tumor cells. Equilibrative nucleoside transporter 2 (ENT2) was previously reported to have the dual ability of transporting both nucleosides and nucleobases. In the present study, we investigated whether or not these nucleobase analogues are transported via ENT2, using mouse ENT2-overexpressing Cos-7 cells. The hypoxanthine uptake mediated by ENT2 was significantly reduced by the addition of 6-MP and 6-TG, and the inhibition of the hypoxanthine uptake by the 6-thiopurines was competitive. Transfection of ENT2 cDNA into Cos-7 cells resulted in an increase in 6-MP uptake. The 6-MP uptake via ENT2 showed clear time- and substrate concentration-dependent profiles, and was inhibited by 6-TG in an inhibitor concentration-dependent fashion. On the other hand, uracil was not a substrate for ENT2, and 5-FU had no effect on the hypoxanthine uptake via ENT2. Therefore, we concluded that 6-MP and 6-TG, but not 5-FU, are transported mediated by the same recognition site on ENT2 with hypoxanthine.