This study introduces a novel method of bovine retinal microcirculation preparation to assess reactivities of retinal microvessels in ex vivo. This preparation is perfused intraluminally through the retinal artery. Effects of vasoactive hormones on these microvessels can be studied by perfusion or topical application to muscle bath. Effects of these compounds on the retinal microvessels of 1A (180- to 200-mu m diameters), 2A (50- to 60-mu m diameter), and 3A (20- to 30-mu m diameter) can be assessed simultaneously using video microscopy. In this study, the water-soluble compound endothelin-l, when applied topically, caused dose-dependent vasoconstriction of all microvessels but had no effect when perfused intraluminally. On the other hand, lipid-soluble prostaglandin F-2 alpha (PGF(2 alpha)) caused dose-dependent vasoconstriction when administered either intra- or extraluminally. Furthermore, preconstricted retinal arterioles (by PGF(alpha)) were dilated in a dose-dependent manner when acetylcholine was perfused through the retinal artery. This vasodilation was attenuated by atropine or N-3-monomethyl arginine, a nitric oxide synthase inhibitor. Topical potassium chloride also caused dose-dependent vasoconstriction of all retinal microvessels. Fluorescein angiography showed no breakdown of the blood-retinal barrier. Thus, in this ex vivo perfused bovine retinal microcirculation preparation, the water-soluble compounds may not cross the blood-retinal barrier to affect microcirculation while lipid soluble can affect this circulation. Additionally this study shows that endothelial-derived compounds, endothelin and nitric oxide, caused vasoconstriction and dilation, respectively, (C) 1994 Academic Press,