Nine C²-substituted adenosine analogues that are potent and selective for the A₂-adenosine receptor were tested for their ability to induce relaxations of the guinea pig aorta. Compounds tested were 2-phenylethoxyadenosine (PEA), 2-phenylethoxy-5′- N-ethylcarboxamidoadenosine (PENECA), 2-cyclohexylethoxyadenosine (CEA), 2-fluorophenylethoxyadenosine (FPEA), 2-methoxyphenylethoxyadenosine (MPEA), 2-naphthylethoxyadenosine (NEA), 2-phenylaminoadenosine (CV-1808), 2-phenylethylaminoadenosine (PEAA) and 2-carboxyethylphenethylamino-5′-N-ethylcarboxamidoadenosine (CGS21680). The responses to these agents were compared to those of three standard adenosine receptor agonists, 5′-N-ethylcarboxamidoadenosine (NECA), N⁶-cyclohexyladenosine (CHA) and R-N⁶-phenylisopropyladenosine (R-PIA). The C²-ethoxyadenosine analogues were 30- to 140-fold less potent than NECA and the C²-amino-substituted analogues were 250 to 1000-fold less potent than NECA at inducing relaxations of the guinea pig aorta. All of the analogues were also less potent than the A₁-selective agonist R-PIA. However, only responses to NECA were competitively antagonized by the non-selective adenosine receptor antagonist 8-phenyltheophylline (8-PT), pK_B=6.83±0.05. The results suggest that the C²-substituted analogues produce relaxations of the guinea pig aorta through a combination of actions at A₂-adenosine receptors and at xanthine resistant sites. The lack of potency of these analogues at activating the xanthine sensitive A₂-receptors in the guinea pig aorta suggests that these adenosine receptors may be of the A_2b-subtype.