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A new method for the spatially resolved measurement of the oxygen saturation of retinal vessels is described. Imaging spectrometry was used for both measurements of transmission and reflectance spectra of whole blood in cuvettes as well as for fundus reflectance spectra. A model was developed for the calculation of the oxygen saturation, valid in the wavelength range between 510 nm and 586 nm, in that the internal reflectance is constant and only the transmitted light depends on layer thickness and hematocrit. Altogether 265 measurements were performed in different number at 30 eyes. In each measurement, the oxygen saturation was simultaneously determined for 193 locations along a line of 1.5 mm at the fundus. The mean oxygen saturation in retinal arteries was (92.2±4.1)% and (57.9±9.9)% in retinal veins. The mean retinal arterio-venous difference of the oxygen saturation was (35.1±9.5)%. The venous oxygen saturation depended on distance from the optic disc. The measured mean of the arterio-venous difference of the oxygen saturation corresponded well to the value of the brain (34%). The utilization of oxygen in the temporal quadrants (inferior: 39.4±10.4%) is significantly (p=0.05) higher than in the nasal quadrants (inferior: 31.3±6.7%).