Levels of cardiovascular active metabolites, like adenosine, are regulated by nucleoside transporters of endothelial cells. We characterized the nucleoside and nucleobase transport capabilities of primary human cardiac microvascular endothelial cells (hMVECs). hMVECs accumulated [³H]2-chloroadenosine via the nitrobenzylmercaptopurine riboside (NBMPR)-sensitive equilibrative nucleoside transporter 1 (ENT1) at a V_max of 3.4 ± 1 pmol µl^-1 s^-1, with no contribution from the NBMPR-insensitive ENT2. Inhibition of 2-chloroadenosine uptake by ENT1 blockers produced monophasic inhibition curves which are also compatible with minimal ENT2 expression. The nucleobase [³H]hypoxanthine was accumulated within hMVECs (Km = 96 ± 37 µM; V_max = 1.6 ± 0.3 pmol µl^-1 s^-1) despite the lack of a known nucleobase transport system. This novel transporter was dipyridamole insensitive but could be inhibited by adenine (K_i = 19 ± 7 µM) and other purine nucleobases including chemotherapeutic analogues. A variety of other cell types also expressed the nucleobase transporter, including the nucleoside transporter deficient cell line PK15NTD. Further characterization of [³H]hypoxanthine uptake in the PK15NTD cells showed no dependence on Na⁺ or H⁺. PK15NTD cells expressing hENT2 accumulated 4.5-fold more [³H]hypoxanthine in the presence of the ENT2 inhibitor dipyridamole than did PK15NTD or hMVEC cells suggesting trapping of ENT2 permeable metabolites. Understanding the nucleoside and nucleobase transporter profile in the vasculature will allow for further study into their roles in patho-physiological conditions such as hypoxia/ischemia.