OBJECTIVE To report an imaging technique for measurement of oxygen tension (PO2) in retinal tissue and establish its feasibility for measuring retinal PO2 variations in rat eyes by adjusting the fraction of inspired oxygen (FiO2). METHODS A narrow laser line was projected at an angle on the retina, and phosphorescence emission was imaged after intravitreal injection of an oxygen-sensitive molecular probe. A frequency-domain approach was used for phosphorescence lifetime measurements. Retinal PO2 maps were computed from phosphorescence lifetime images, and oxygen profiles through the retinal depth were derived in rats in conditions of 10%, 21%, and 50% FiO2. RESULTS Retinal PO2 measurements were repeatable, and variations in outer and inner retina PO2 at different locations along the image were not significant (P>or=0.3). Maximum outer retinal PO2 obtained in 10%, 21%, and 50% FiO2 were significantly different (P<0.0001). Maximum outer retinal PO2 correlated with systemic arterial PO2 (R=0.70; P<0.0001). The slope of the outer retina PO2 profile correlated with maximum outer retinal PO2 (R=0.84; P<0.0001). Mean inner retina PO2 correlated with maximum outer retinal PO2 (R=0.88; P<0.0001). CONCLUSIONS A technique has been developed for quantitative mapping of retinal tissue oxygen tension with the potential to enable sequential monitoring of retinal oxygenation in health and disease.