The human and rat equilibrative nucleoside transporter proteins hENT1, rENT1, hENT2 and rENT2 belong to a family of integral membrane proteins with 11 potential transmembrane segments (TMs), and are distinguished functionally by differences in transport of nucleobases and sensitivity to inhibition by nitrobenzylthioinosine (NBMPR) and vasoactive drugs. In the present study, we have produced recombinant hENT1, rENT1, hENT2 and rENT2 in Xenopus oocytes and investigated uridine transport following exposure to the impermeant thiol-reactive reagent p-chloromercuriphenyl sulphonate (PCMBS). PCMBS caused reversible inhibition of uridine influx by rENT2, but had no effect on hENT1, hENT2 or rENT1. This difference correlated with the presence in rENT2 of a unique Cys residue (Cys(140)) in the outer half of TM4 that was absent from the other ENTs. Mutation of Cys(140) to Ser produced a functional protein (rENT2/C140S) that was insensitive to inhibition by PCMBS, identifying Cys(140) as the exofacial Cys residue in rENT2 responsible for PCMBS inhibition. Uridine protected wild-type rENT2 against PCMBS inhibition, suggesting that Cys(140) in TM4 lies within or is closely adjacent to the substrate-translocation channel of the transporter. TM4 has been shown previously to be within a structural domain (TMs 3-6) responsible for interactions with NBMPR, vasoactive drugs and nucleobases.