Adenosine A_2B receptors are known as low-affinity receptors due to their modest-to-negligible affinity for adenosine and prototypic agonists. Despite numerous synthetic efforts, 5′-N-ethylcarboxamidoadenosine (NECA) still is the reference agonist, albeit nonselective for this receptor. In our search for higher affinity agonists, we developed decision schemes to select amino acids for mutation to the corresponding residues in the most homologous, higher affinity, human A_2A receptor. One scheme exploited knowledge on sequence alignments and modeling data and yielded three residues, V11, L58, and F59, mutation of which did not affect agonist affinity. The second scheme combined knowledge on sequence alignments and mutation data and pointed to Ala12 and Asn273. Mutation of Ala12 to threonine did not affect the affinity for NECA, (R)-N ⁶-(phenylisopropyl)adenosine (R-PIA), and 2Cl Ado. The affinity of the N273Y mutant for NECA and R-PIA and for the antagonists xanthine amine congener (XAC), ZM241385, and SCH58261 was also unaltered. However, this mutant had a slightly increased affinity for a 2-substituted adenosine derivative, CGS21680. This prompted us to investigate other 2-substituted adenosines, with selectivity and high affinity for A_2A receptors. All four compounds tested had improved affinity for the N273Y receptor. Of these, 2-(1-hexynyl)adenosine had submicromolar affinity for the N273Y receptor, 0.18 ± 0.10 μM, with a 61-fold affinity gain over the wt receptor. In addition, the non-NECA analog (S)-PHP adenosine had an affinity of 1.7 ± 0.5 μM for the wt receptor. The high affinity of (S)-PHP adenosine for the wt receptor suggests that further modifications at the 2-position may yield agonists with even higher affinity for A_2B receptors.