In order to relate optical measurement of the retinal nerve fiber layer (RNFL) to the underlying structure, one must have accurate values for RNFL thickness at the locations measured optically. The purpose of this study was to develop a method for measuring RNFL thickness at any location on retinal tissue previously studied by other optical imaging. The method developed used confocal laser scanning microscopy (cLSM) to provide both en face and cross-sectional images of a whole-mounted retina. Isolated rat retina was fixed with 3% glutaraldehyde. Nerve fiber bundles were identified by using phalloidin to label F-actin and ganglion cell bodies were identified by DAPI fluorescent counterstain of nuclei. The flat-mounted retina was examined by cLSM. 2-D images were collected through the retina to a depth at least covering the ganglion cell layer. The images were stacked to reconstruct cross-sectional images of the measured retina. Thickness of nerve fiber bundles was measured on these synthesized cross sections and compared with the measurement from conventional histologic sections. The en face image displayed individual nerve fiber bundles and ganglion cells between bundles as different colors. Blood vessels, which also bound phalloidin, were easily distinguished from nerve fiber bundles. The en face image displayed the same pattern of nerve fiber bundles as seen in imaging measurements and simplified the identification of corresponding areas in the two modalities. The cross-sectional images provided thickness measurements of the RNFL over the entire field-of-view, not just at the points represented by the conventional histologic section, resulting in a large increase in available data.