Scanning laser polarimetry (SLP) assesses the retinal nerve fiber layer (RNFL) for glaucoma diagnosis by detecting the birefringence of the peripapillary RNFL. A detailed understanding of SLP requires an accurate value for RNFL birefringence in order to relate measured retardance to RNFL thickness, but current knowledge of this value is limited. A multispectral imaging micropolarimeter of PSC′A type was used to measure the retardance in transmission of the RNFL of isolated rat retina before (living) and after (fixed) 20 min of glutaraldehyde fixation. The thickness of the nerve fiber bundles measured was then determined histologically. As previously known from reflectance measurements, in transmission the RNFL behaved as a linear retarder. The retardance of the RNFL was constant at wavelengths from 440 to 830 nm and persisted after tissue fixation. In 37 nerve fiber bundles of 8 retinas, the average RNFL birefringence was 0.23 nm/μm before and 0.19 nm/μm after fixation, with an uncertainty of 0.01 nm/μm. The wavelength independence is consistent with a mechanism of form birefringence from thin cylindrical organelles. These results allow extrapolation of previous visible wavelength measurements to the near-infrared wavelengths used by SLP and validate the use of fixed tissue for RNFL research. © 2002 Society of Photo-Optical Instrumentation Engineers.