The oxygen tension in the vessels of the retina and optic nerve head has been measured noninvasively with a new phosphorescence imaging method. A phosphorescent oxygen-dependent probe, injected into the bloodstream of cats, was excited with a flash of light and the phosphorescence lifetime of the probe was measured. A simple Stern-Volmer relationship was used to convert lifetime to oxygen tension, and two-dimensional maps of intravascular oxygen tension were produced. We describe the equipment and the methodology for obtaining oxygen maps.