Nucleoside transporter inhibitors have potential therapeutic applications as anticancer, antiviral, cardioprotective, and neuroprotective agents. We have synthesized and flow cytometrically evaluated the binding affinity of a series of novel halogenated nitrobenzylthioinosine analogs at the human es nucleoside transporter. Structure-activity relationships indicate the importance of hydrophobicity and electron withdrawing capacity of substituents at the para-position of the 6-position benzyl substituent. All of the compounds showed high binding affinity as shown by their ability to displace the fluorescent es transporter ligand, SAENTA-X8-fluorescein. Compound 16 (6-S-(para-iodobenzyl)-6-thioinosine) was the most tightly bound within the series with a K(i) of 3.88 nM (NBMPR exhibited a K(i) of 0.70 nM). This compound has higher affinity than the widely used nonnucleoside, nucleoside transport inhibitor, dipyridamole (K(i) = 8.79 nM), and may serve as a new lead compound.