The reactivity of retinal arterioles and venules to exogenous and endogenous adenosine was investigated in the newborn piglet eye in vivo. The retinal microcirculation of isoflurane-anesthetized newborn pigs was observed at 310× using videomicroscopy, and changes in the diameter of arterioles (50-100 μm in diameter) and venules (150-250 μm in diameter) occurring in response to intravitreal topical microsuffusions of various adenosi-nergic compounds were determined. Dose-dependent dilations of the arterioles and venules resulted from intravitreal adenosine (0.2-200 nmol) and three of its agonists: 5'-N-ethylcarboxyamidoadenosine (0.2-20 nmol), 2-chloroaden-osine (0.2 pmol-2 nmol), and N6-cyclohexyladenosine (0.2 and 2.0 nmol). The resulting order of vasodilatative potency, wherein 5'-N-ethylcarboxyamidoadenosine >> 2-chloroadenosine > adenosine = N6-cyclohexyladenosine, is indicative of A2 adenosine receptor-mediated vasodilation. Significant arteriolar dilations [24 ± 4% (p = 0.0012) and 35 ± 5% (p = 0.0008), respectively) also resulted from intravitreal application of 0.2 nmol of iodotubercidin, an adenosine kinase inhibitor, and 0.1 nmol of nitrobenzyl-thioinosine, an inhibitor of adenosine reuptake/transport. The dilation induced by nitrobenzylthioinosine was blocked (p < 0.0004) by coadministration of the relatively specific A2 adenosine receptor antagonist l,3-dipropyl-7-methylxanthine (1 nmol), confirming that nitrobenzylthioinosine induced dilation via potentiation of endogenous adenosine. Administration of l,3-dipropyl-7-methylxan-thine alone did not significantly affect arteriolar or venular diameters, suggesting that endogenous adenosine does not contribute to the maintenance of basal tone. Collectively, these results indicate that vascular A2 adenosine receptors mediate retinal arteriolar dilation in response to increases in endogenous adenosine concentrations, consistent with our hypothesis that endogenous interstitial adenosine plays an important role in the metabolic regulation of retinal blood flow.