A series of 55 2-alkyloxy-, 2-aryloxy- and 2-aralkyloxy-adenosines was screened as inhibitors of the binding of [3H]R-phenyl-isopropyladenosine to A1 adenosine receptors in rat cerebral cortical membranes, and of the binding of N-ethylcarboxamidoadenosine to A2 adenosine receptors in rat striatal membranes and as agonists at A2 adenosine receptors coupled to adenylate cyclase in rat pheochromocytoma PC12 cell membranes. The activities are consonant with a hydrophobic binding site in the A2 receptors at a distance from the 2-position of the adenine ring corresponding to a spacer chain of -O-CH2-CH2-. These is little lateral steric tolerance in the region occupied by the spacer chain. Interaction with the hydrophobic binding site is greatest in the 2-alkyloxy series for 2-cyclohexylethoxy-, 2-cyclohexylpropoxy- and 2-cyclohexylbutoxyadenosines and in the 2-aralkoxy series for 2-phenylethoxy-, 2-(4-methylphenyl)ethoxy-, 2-(4-chlorophenyl)ethoxy-, and 2-naphthylethoxy-adenosine. The affinities of the 2-substituted adenosines for the rat cerebral cortical A1 receptors are not as markedly altered by structural changes and are in almost all cases two- to hundredfold less than the affinity of the 2-substituted adenosine for the rat striatal A2 receptor. There is excellent correspondence of the present data on rat A2 receptors with reported potencies of these 2-substituted adenosines as coronary vasodilators in guinea pig heart preparations.