Nucleoside and nucleobase transporters are important for salvage of purines and pyrimidines and for transport of their analog drugs into cells. However, the pathways for nucleobase translocation in mammalian cells are not well characterized. We identified a Na-independent purine-selective nucleobase/nucleoside transport system in the nucleoside transporter deficient PK15NTD cells. This transport system has 1000-fold higher affinity for nucleobases than nucleosides with K_m values of 2.5±0.7 µM for [³H]adenine, 6.4±0.5 µM for [³H]guanine, 1.1±0.1 mM for [³H]guanosine and 4.2±0.5 mM [³H]adenosine. The uptake of [³H]guanine (0.05 µM) was inhibited by other nucleobases and nucleobase analog drugs (at 0.5-1mM in the order of potency): 6-mercaptopurine = thioguanine = guanine > adenine >>> thymine = fluorouracil = uracil. Cytosine and methylcytosine had no effect. Nucleoside analog drugs with modification at 2’ and/or 5 positions (all at 1mM) were more potent than adenosine in competing the uptake of [³H]guanine: 2-chloro-2’-deoxyadenosine > 2-chloroadenosine > 2’3’-dideoxyadenosine = 2-deoxyadenosine > 5-deoxyadenosine > adenosine. 2-chloro-2’-deoxyadenosine and 2-chloroadenosine inhibited [³H]guanine uptake with IC₅₀ values of 68±5 and 99±10µM, respectively. The nucleobase/nucleoside transporter was resistant to NBMPR, dipyridamole and dilazep, but was inhibited by papaverine, the organic cation transporter inhibitor, decynium-22 (IC₅₀ ~1 µM) and by acidic pH (pH=5.5). In conclusion, we have identified a mammalian purine-selective nucleobase/nucleoside transporter with high affinity for purine nucleobases. This transporter is potentially important for transporting naturally occurring purines and purine analog drugs into cells.