The goal of making calibrated oxygen saturation measurements of blood in retinal arteries and veins via a noninvasive spectroscopic technique has nearly been realized. Semi-continuous advancement in the field of retinal vessel oximetry over the last three decades has resulted in several technologies that seem poised for commercialization. In this paper, we present our instrumentation and technique for making well-calibrated saturation measurements of the blood in retinal vessels. The Eye Oximeter (EOX) is a confocal scanning laser ophthalmoscope capable of acquiring multi-spectral images. Analysis of these spectral vessel images allows spectroscopic determination of the oxygen saturation of blood within each vessel. The primary emphasis of this paper is to illustrate the effect of fundus pigmentation on these oximetric measurements. We show that decreasing fundus reflectivity is mathematically similar to decreasing the vessel thickness. The apparent decreased vessel thickness is a direct consequence of scattering by red blood cells. We present in vitro and in vivo measurements that demonstrate an instrument calibration that is nearly independent of vessel diameter and fundus reflectivity.© (2001) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.