Human equilibrative nucleoside transporters 1 and 2 (hENT1 and hENT2) differ functionally in that hENT2 generally displays lower affinity for its nucleoside permeants and is less sensitive to inhibition by the coronary vasodilators dilazep and dipyridamole. In previous work, we demonstrated that mutation of residues 33 (Met versus Ile) of hENT1 and hENT2 altered sensitivity to dilazep and dipyridamole and that the hENT2 mutant (I33M) displayed a K(m) value for uridine that was lower than that of hENT2 and similar to that of hENT1 (J Biol Chem 277:395-401, 2002). In this study, we report results of an in-depth investigation of the role of residue 33 in hENT2. We found that hENT2-I33M displayed decreased K(m) values for both pyrimidine and purine nucleosides and increased V(max) values for purine nucleosides. Cys or Ser at position 33 had similar effects on the kinetic parameters of hENT2 as Met, indicating that hydrophobic (Met and Cys) or hydrogen-bonding energy (Ser) contributed to permeant binding by these residues. hENT2-I33M and I33C displayed increased sensitivities to dipyridamole compared with wild-type hENT2, hENT2-I33A, and hENT2-I33S, suggesting interaction of the sulfur atom of Met and Cys with aromatic moieties on dipyridamole. hENT2-I33C was inhibited by the membrane-impermeant sulfhydryl reactive reagent p-chloromercuribenzyl sulfonate, and uridine, adenosine, and dipyridamole protected against inhibition. Our results indicated that residue 33 resides in an extracellular domain as predicted by the current hENT2 topology model and suggested that it is a functionally important component of both the permeant and dipyridamole binding sites.