A series of 2-phenylethynyladenosine (PEAdo) derivatives substituted in the N⁶- and 4'-position was synthesised and the new derivatives were tested at the four human adenosine receptors stably transfected into Chinese hamster ovary (CHO) cells, using radioligand binding studies (A₁, A(₂A), A₃) or adenylyl cyclase activity assay (A(₂B)). Binding studies showed that the presence of a phenyl ethynyl group in the 2 position of adenosine favoured the interaction with A₃ receptors, resulting in compounds endowed with high affinity and selectivity for the A₃ subtype. Additional substitution of the N⁶- and 4'-position increases both A₃ affinity and selectivity. The results showed that the new compounds have a good affinity for the A₃ receptor and in particular, the N⁶-methoxy-2-phenylethynyl-5'-N-methylcarboxamidoadenosine, with a K(i) at A₃ of 1.9 nM and a selectivity A₁/A₃ and A(₂A)/A₃ of 4,800- and 8,600-fold, respectively. Therefore, it is one of the most potent and selective agonists at the human A₃ adenosine receptor subtype reported so far. Furthermore, functional assays of inhibition of 10 μM forskolin-stimulated cAMP production via the adenosine A₃ receptor revealed that the new trisubstituted adenosine derivatives behave as full agonist of this receptor subtype. Docking analysis of these compounds was performed at a homology model of the human A₃ receptor based on the bovine rhodopsin crystal structure as template, and the results are in accordance with the biological data.