This paper reports the first quantitative measurements of the reflectance spectrum of the nerve fiber layer (NFL) of the retina. An arcuate defect in the NFL of two macaque monkeys was produced by photocoagulating a small spot near the optic disc. Imaging fundus reflectometry at 8 wavelengths was then used to measure the fundal reflectances in the defect and in the intact NFL. The reflectance of the NFL alone was determined by subtracting the reflectance of the bare fundus in the defect from the reflectance of the adjoining fundus with intact NFL. The reflectance data were corrected for the transmission of the ocular media with published values of the density of monkey cornea and lens. Lens absorption was measured from Purkinje images and differed little from the published values. The NFL reflectance can be characterized by a spectrum that has the same shape at all points along an arcuate region of the NFL but decreases in absolute reflectance as the thickness of the NFL decreases. The reflectance changes little between 680 and 560 nm, but rises as lambda-n from 560 to 460 nm, where n = 2.5-3.1. The data reported here begin to provide a quantitative understanding of empirically determined methods for enhancing the visibility of the NFL and have implications for retinal densitometry.