When supplementing lamb diets with vitamin E, an equivalence factor of 1.36 is used to discriminate between RRR-α-tocopheryl acetate and all-rac-α-tocopheryl acetate. However, more recent studies suggest a need for new equivalence factors for livestock animals. The current study aimed to determine the effect of RRR- and all-rac-α-tocopheryl acetate supplementation on α-tocopherol deposition in lamb tissues. A total of 108 Rasa Aragonesa breed lambs were fed increasing amounts of all-rac-α-tocopheryl acetate (0.25, 0.5, 1.0 and 2.0 g/kg compound feed) or RRR-α-tocopheryl acetate (0.125, 0.25, 0.5 and 1.0 g/kg compound feed) by adding them to a basal diet that contained 0.025 g/kg feed of all-rac-α-tocopheryl acetate as part of the standard vitamin and mineral mixture. The diets were fed for the last 14 days before slaughtering at 25.8±1.67 kg BW. Within 20 min after slaughter samples of muscle, heart, liver, brain and spleen were frozen at −20°C until α-tocopherol analysis. Increased supplementation of either vitamin E sources led to a significant increase (P<0.001) in α-tocopherol concentration in all tissues studied. The tissue with the highest α-tocopherol concentration was the liver followed by spleen, heart and muscle. At similar supplementation levels (0.25, 0.50 and 1.0 g/kg compound feed), α-tocopherol content in the selected tissues was not affected by α-tocopherol source. However, the ratios between RRR- and all-rac-α-tocopheryl acetate increased with the increasing α-tocopherol supplementation (at 0.25 and 1.0 g/kg compound feed), from 1.06 to 1.16 in muscle, 1.07 to 1.15 in heart, 0.91 to 0.94 in liver and 0.98 to 1.10 in spleen. The highest relative proportion of Ʃ2S (sum of SSS-, SSR-, SRS- and SRR-α-tocopherol)-configured stereoisomers was found in the liver of lambs supplemented with all-rac-α-tocopheryl acetate accounting for up to 35 to 39% of the total α-tocopherol retained, whereas the proportion of Ʃ2S-configured stereoisomers in the other tissues accounted for <14%. Increasing all-rac-α-tocopheryl acetate supplementation was also found to affect the 2R-configured stereoisomer profile in muscle, heart and spleen with increasing proportions of RRS-, RSR- and RSS- at the cost of RRR-α-tocopherol. In all tissues, the relative proportion of all non-RRR-stereoisomers in lambs receiving RRR-α-tocopheryl acetate was lower than RRR-α-tocopherol. These results confirm that the relative bioavailability of RRR- and all-rac-α-tocopheryl acetate is dose- and tissue-dependent and that a single ratio to discriminate the two sources cannot be used.