A number of 2-substituted 5'-N-ethylcarboxamidoadenosine (NECA) derivatives was investigated for their affinity and selectivity at human A3 adenosine receptors. The compounds were tested in radioligand competition studies and modulation of adenylyl cyclase activity on membranes from CHO cell lines stably transfected with the four human adenosine receptor subtypes. In binding studies the most potent compound, 2-(3-hydroxy-3-phenyl)propyn-1-yl-NECA (PHPNECA), exhibited a subnanomolar affinity for A3 adenosine receptors with a Ki value of 0.4 nM. As opposed to the limited A3 selectivity of PHPNECA, a 100-fold selectivity compared to both A1 and A2A receptors was found for 2-(2-phenyl)ethynyl-NECA (PENECA; Ki 6 nM). The EC50 values for activation of adenylyl cyclase via A2A adenosine receptors were in good agreement with the respective Ki values from binding experiments. In contrast, IC50 values for A1 and A3 receptor-mediated inhibition of adenylyl cyclase were shifted to higher values compared to the respective affinities determined in radioligand competition studies. Similar discrepancies between binding and functional data have been observed for the inhibitory A1 adenosine receptor in previous studies. Therefore, the same A3 selectivity of PENECA compared to A1 receptors was found in binding and adenylyl cyclase inhibition whereas the selectivity compared to A2A receptors that was detected in ligand binding was obscured in the functional assay. The series of compounds presented in this study identifies 2-substitution of the purine system as a promising target for the development of A3-selective high-affinity ligands.