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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 K _i value of 0.4 nM. As opposed to the limited A₃ selectivity of PHPNECA, a 100-fold selectivity compared to both A₁ and A_2A receptors was found for 2-(2-phenyl)ethynyl-NECA (PENECA; K _i 6 nM). The EC₅₀ values for activation of adenylyl cyclase via A_2A adenosine receptors were in good agreement with the respective K _i 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 A_2A 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. [ABSTRACT FROM AUTHOR]