Vitamin E requirements of adult Standardbred horses were evaluated by tissue depletion and repletion. All the horses used in the study were given the same basal feed low in vitamin E during the eight months of the experiment. After an initial depletion period of two-and-a-half months the horses were divided into groups according to the amounts of DL α-tocopheryl acetate given (0 mg, control; 200, 600, 1800 and 5400 mg, respectively) as a daily oral supplement. The supplement study was followed by a second depletion period. Total vitamin E content and individual natural tocopherol isomers and tocotrienol isomers were measured both in the feed (hay and oats) and in tissues (serum, liver, skeletal muscle and adipose tissue) using high performance liquid chromatography with fluorescent detection. Tissue vitamin E response to different dietary vitamin E levels were studied. The serum total lipid content remained unchanged during the experiment; serum vitamin E levels were expressed per gram serum lipid. The total vitamin E levels in serum, liver, skeletal muscle and fat reflected the supplement levels. The highest vitamin E levels were seen in fat tissue, followed by the liver and by skeletal muscle. In spite of the wide occurrence of the different vitamin E isomers in the feed, α-tocopherol was almost the only isomer detected in the tissues. To ensure nutritional adequacy, 600 and 1800 mg of dl α-tocopheryl acetate was suggested as an optimal oral daily supplement of vitamin E to adult Standardbred horses given feed low in vitamin E; this corresponds to 1.5 to 4.4 mg/kg bodyweight. In addition, it appeared preferable to give the supplement daily to maintain a constant level of vitamin E in the serum, liver and skeletal muscle, rather than occasionally to give higher doses.