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Site-directed mutagenesis was used to target residues within transmembrane domains 1 and 2 of the human equilibrative nucleoside transporter 1 (hENTl), in order to assess their contributions to transporter functionality. Through affinity changes in [ H]NBMPR binding, competitive inhibition profiles with ENT1 inhibitors and [ H]2- chloroadenosine uptake, these residues were found to make minor, but statistically significant, contributions to the inhibitor and/or substrate binding site of hENTl. An initial study was also undertaken to probe the potential oligomerization of hENTl. Typically considered to be a monomer to this date, under the native conditions of Blue Native Electrophoresis, hENTl was found to possess a molecular mass of 160 kDa. This is approximately three times the size of the known 55kDa hENTl monomer. Targeted site-directed mutagenesis of GxxxG motifs within the transporter, known to play a role in oligomerization, did not appear to affect the higher molecular mass hENTl complex observed