The effect of supplementation with different levels of all-rac-α-tocopheryl acetate and the inclusion of different dietary contents of PUFA on the deposition of α-tocopherol stereoisomers in liver and thigh of chickens was evaluated. Ninety-six 1-d-old Ross female broiler chickens were randomly distributed into eight experimental treatments (three replicates each) resulting from four levels of α-tocopheryl acetate without supplementation and supplemented with 100, 200 and 400 mg α-tocopheryl acetate/kg and two levels of dietary PUFA (15 and 61 g/kg). The feeds supplemented with α-tocopheryl acetate contained a similar proportion of each stereoisomer. The diets without α-tocopheryl acetate had the following α-tocopherol stereoisomers (%): RRR 35·1, RRS 24·5, RSR 25·3, RSS 13·9 and total 2S forms 1·3. Consumption of different levels of α-tocopheryl acetate did not lead to statistical differences in α-tocopherol stereoisomer proportion in the liver and thigh. In general, the stereoisomer profiles in the tissues studied were similar, responding to the stereoisomer profile of the diet. Both tissues preferentially accumulated 2R stereoisomer (69–100%). However, when α-tocopheryl acetate was used the discrimination was not specific for the RRR α-tocopherol form. Furthermore, the 2R:2S ratio had a tendency to increase as the polyunsaturation level of the diet increased.