The diattenuation spectrum of the retinal nerve fiber layer (RNFL) reflectance has been predicted to depend strongly on the relative refractive index (m) of light-scattering cylinders. To constrain the values of m, diattenuation of the RNFL reflectance of isolated rat retina was measured with a multispectral imaging micropolarimeter. The RNFL reflection has very weak intrinsic diattenuation at all wavelengths (400-830 nm), which rejects all values of m > or = 1.03. Degree of polarization (DOP) for reflection from the RNFL was also measured. DOP was close to unity at all wavelengths, which indicates that the RNFL is a polarization-preserving reflector.