hENT1 and hENT2 are members of the human equilibrative nucleoside transporter family. hENT1 is ubiquitously expressed and plays an important role in the disposition and pharmacological activity of nucleoside drugs and nucleosides, such as adenosine. hENT2 is expressed in only a few tissues (e.g. muscle). hENT1 and hENT2 differ in their affinity for nucleoside substrates and in their sensitivity to inhibitors, such as nitrobenzylthioinosine (NBMPR). hENT1 has higher (or equal) affinity to hENT2 for all natural nucleosides except inosine. hENT1 is also more sensitive to NBMPR inhibition (IC50 approximately 0.4-8 nM) when compared with hENT2 (IC50 approximately 2.8 microM). This difference in inhibition potency is substantially dependent on the difference in amino acid at position 154 in hENT1 (glycine) and hENT2 (serine). Since NBMPR competitively inhibits nucleoside transporter activity, we hypothesized that G154 may also play a role in the transport of natural nucleosides and in the inhibition by other hENT1 inhibitors, dipyridamole (DP), and dilazep (DZ). Our results, using a yeast expression system, demonstrate that substituting glycine 154 of hENT1 with serine of hENT2 converts hENT1 to a transporter that exhibits partial characteristics of hENT2. For example, this conversion reduces sensitivity of hENT1 to the inhibitors NBMPR, DP, and DZ and reduces its transport affinity for the natural nucleosides cytidine and adenosine. However, this conversion renders hENT1 less sensitive to inhibition by anti-HIV drugs azidothymidine, dideoxyinosine, and the nucleobase, hypoxanthine. Collectively, these results suggest that glycine 154 plays an important role in the transport of nucleosides and in sensitivity to the inhibitors NBMPR, DP, and DZ.