1  James E. Garrette Eye Research Laboratory, Department of Ophthalmology and 2  Department of Anesthesiology, University of Virginia Health Sciences Center, Charlottesville, Virginia 22903 A method for noninvasive measurement of Hb O 2 saturation (S O 2 ) in retinal blood vessels by digital imaging was developed and tested. Images of vessels were recorded at O 2 -sensitive and O 2 -insensitive wavelengths (600 and 569 nm, respectively) by using a modified fundus camera with an image splitter coupled to an 18-bit digital camera. Retinal arterial S O 2 was varied experimentally by having subjects breathe mixtures of O 2 and N 2 while systemic arterial S O 2 was monitored with a pulse oximeter. Optical densities (ODs) of vascular segments were determined using a computer algorithm to track the path of reflected light intensity along vessels. During graded hypoxia the OD ratio (ODR = OD 600 /OD 569 ) bore an inverse linear relationship to systemic S O 2 . Compensation for the influence of choroidal pigmentation significantly reduced variation in the arterial S O 2 measurements among subjects. An O 2 sensitivity of 0.00504 ± 0.00029 (SE) ODR units/%S O 2 was determined. Retinal venous S O 2 at normoxia was 55 ± 3.38% (SE). Breathing 100% O 2 increased venous S O 2 by 19.2 ± 2.9%. This technique, when combined with blood flow studies in human subjects, will enable the study of retinal O 2 utilization under experimental and various disease conditions. hemoglobin oxygen saturation; spectrophotometry