OBJECTIVE To measure and describe the reflectance properties of a mammalian retinal nerve fiber layer (RNFL) and to determine the mechanisms responsible for the RNFL reflectance. METHODS An isolated rat retina suspended across a slit in a black membrane and mounted in a black perfusion chamber provided high quality images of the RNFL. Imaging microreflectometry was used to measure RNFL reflectance at wavelengths from 400 nm to 830 nm and as a function of illumination angle. RESULTS The directional reflectance of rat RNFL at all wavelengths was consistent with the theory of light scattering by cylinders; each nerve fiber bundle scattered light into a conical sheet coaxial with the bundle. There was no evidence of a noncylindrical component at any wavelength. Measured reflectance spectra were consistent between animals, similar to ones previously measured in macaque, and varied with scattering angle. All spectra could be described by a two-mechanism cylindrical scattering model with three free parameters. CONCLUSIONS At all wavelengths the reflectance of rat RNFL arises from light scattering by cylindrical structures. The highly directional nature of this reflectance can be an important source of measurement variability in clinical assessment of the RNFL. The reflectance spectra reveal a combination of mechanisms: At wavelengths shorter than approximately 570 nm the reflectance comes from cylinders with diameters much smaller than the wavelength, but at wavelengths longer than approximately 680 nm the reflectance comes from cylinders with effective diameters of 350 nm to 900 nm.