Autofluorescence from reduced pyridine nucleotides (PN) and oxidized flavoproteins (Fp) was measured in order to detect the difference in redox states in rabbit corneal epithelium. The enucleated rabbit eye was mounted in an eye bank eye container with McCarey-Kaufman medium, and the autofluorescence was measured using ocular redox fluorometry as a function of depth. The PN signal distributed evenly whereas the Fp signal was greater in the posterior epithelial region than in the anterior region (p < 0.05). The PN/Fp ratio, a sensitive indicator of tissue redox state, was less in the posterior region. After the application of 1 mM of potassium cyanide in the medium, the ratio increased significantly in each layer (p < 0.001), and the difference between anterior and posterior region diminished. These results indicate that ocular redox fluorometry has the potential to resolve the redox states of the various layers of the corneal epithelium. The posterior region of the epithelium is more active in mitochondrial respiration than the anterior region.