Inhibitor and substrate interactions with equilibrative nucleoside transporter 1 (ENT1; SLC29A1) are known to be affected by cysteine-modifying reagents. Given that selective ENT1 inhibitors, such as nitrobenzylmercaptopurine riboside (NBMPR), bind to the N-terminal half of the ENT1 protein, we hypothesized that one or more of the four cysteine residues in this region were contributing to the effects of the sulfhydryl modifiers. Recombinant human ENT1 (hENT1), and the four cysteine-serine ENT1 mutants, were expressed in nucleoside transport-deficient PK15 cells and probed with a series of methanethiosulfonate (MTS) 3 H]2-chloroadenosine transport. The results of this study also indicate that the hENT1-C193S mutant may be useful as a MTSET/MTSESinsensitive transporter for future cysteine substitution studies to define the extracellular domains contributing to the binding of substrates and inhibitors to this critical membrane transporter.