A series of new 2-(ar)alkenyl, both Z- and E-diastereomers, and 2-alkyl derivatives of adenosine-5'-N-ethyluronamide (NECA) and adenosine were synthesized and evaluated for their interaction with the A1 and A2A adeosine receptors, to better understand the conformational requirements of the receptor area interacting with the substituents in the 2- and 5'-positions. Partial reduction of the triple bond in 2-alkynyl derivatives of NECA led to compounds whose activity at the A2A receptor subtype was related to Z-E-isomerism, the E-diastereomers being more potent and selective than the Z-ones. Saturation of the side chain markedly reduced compound affinity at adenosine receptors. Specifically, compounds bearing an (E)-alkenyl chain, while maintaining the same affinity at A2A receptors as the corresponding alknyl derivatives, showed an increase in A2A vs A1 selectivity. Hence, the new nucleosides (E)-2-hexenylNECA (12a) and (E)-2-(phenylpentenyl)NECA (12b) exhibited both high A2A receptor affinity (Ki = 1.6 and 3.5 nM, respectively) and A2A vs A1 selectivity (157- and 290-fold, respectively). Moreover, 12a displayed potent antiaggregatory activity, similar to that of the reference compound NECA. Comparison between NECA and adenosine derivatives further demonstrated that the 5'-ethylcarboxamido group is critical for the A2A affinity. These studies indicated that the orientation of the substituent in the 2-position and the nature of the 5'-group in adenosine derivatives are critical to achieve high affinity and selectivity at the A2A adenosine receptor subtype.