Various optical methods for assessing the retinal nerve fiber layer (RNFL) depend on reflected light, but little is known about the characteristics of the RNFL as a reflecting structure. The authors investigated the angular dependence of light reflected by~flie unmyelinated nerve fibers of the toad eyecup using a small 500-nm light source that could illuminate the retina from various directions and a movable low-power microscope that imaged the retina onto a cooled charge-coupled device in a digital camera system. Measured areas had nerve fiber bundles separated by gaps. Therefore, the reflectance of a bundle alone could be determined from the difference in intensity between the bundle and an adjacent gap. The RNFL reflectance showed striking directional dependence; nerve fiber bundles seen when illuminated from one direction disappeared completely when illuminated from another. Light reflected by a bundle was confined to a conical sheet concentric with the axis of the bundle. The apex angle of the cone was twice the angle between the incident light and the bundle axis, and the orientation of the cone changed with the orientation of the RNFL. This behavior was consistent with the theory of light scattering by cylinders. Therefore, it was concluded that the RNFL reflectance arises from cylindric structures. These results have clinical significance for imaging the RNFL in the human eye because the apparent intensity of the RNFL will depend, not just on its thickness, but also on its