Vitamin E is thought to be important for protection of polyunsaturated fatty acids (PUFA) from oxidative damage. A microbiochemical procedure using microdissection and gas chromatography-mass spectrometry was developed to determine vitamin E distribution in ocular tissues in a rodent model, with the eventual goal of using it in a study of phototoxic degeneration of the retina, where PUFA oxidation is potentially the causal mechanism. Sample preparation was achieved by freeze-drying the retina followed by micro-dissection to obtain the desired structures for analysis. A deuterated alpha-tocopherol internal standard is added to the tissue sample before extraction and derivatization which are achieved in a single step. The data presented show the vitamin-E content in various structures of the retina, particularly the outer segments and retinal pigment epithelium (RPE); however, the vitamin E content of other ocular tissues is also included. Data were obtained from albino and pigmented rats receiving vitamin E-depleted, supplemented, and regular chow diets, and from rabbits and cats receiving regular chow diets formulated for each species. Within all dietary groups the highest concentration of vitamin E was located in the RPE followed by the outer segments of the photoreceptor cells. Other ocular tissues consistently contained lower amounts of vitamin E. Different tissues were depleted of vitamin E at different rates and this points out the importance of determining vitamin E levels in tissues of interest in studies on the consequences of dietary depletion.