Milk fat globule membranes (MFGM) were isolated from milk from cows injected intraperitoneally with dl-α-tocopherol acetate. The fatty acid composition and content, and the contents of tocopherols, retinol and β-carotene were determined and compared with the composition and content in the original cream sample. Intraperitoneal injection of 10 g dl-α-tocopherol acetate elevated the α-tocopherol content in the milk fat from 13–30 to 50–70 εg α-tocopherol/g milk fat 2–3 d after injection. The increase depended on the α-tocopherol status of the cow prior to injection. The concentrations of retinol and β-carotene in the milk fat were unchanged after the α-tocopherol injections. MFGM fatty acids made up 18–27 g/kg total fatty acids in the milk fat. However, the proportions of monounsaturated and polyunsaturated fatty acids were higher in MFGM than in total milk fat, while the proportion of saturated fatty acids was lower in MFGM (P < 0·001). Thus, the longchain polyunsaturated fatty acids in MFGM constituted ∼ 40–70 g/kg total milk fat. α-Tocopherol was the only fat-soluble vitamin detected in MFGM; γ-tocopherol, retinol and β-carotene were detected only in the cream. A significant relationship between α-tocopherol contents in cream and MFGM was found: (α-tocopherol in MFGM fatty acids) =–1652 × (1/ln(α-tocopherol in cream fatty acids)3)+ 97 (r = 0·857, P < 0·001) when the contents of α-tocopherol were expressed as εg/g fatty acid. According to this equation the upper limit for incorporation of α-tocopherol into MFGM is 97±5 εg/g MFGM fatty acids. Thus, under normal farming conditions the α-tocopherol content will be highest in MFGM. However, in cream with an α-tocopherol concentration < 15 εg/g cream fatty acids the concentration in MFGM will be lower and furthermore will decrease rapidly. The possible importance of this relationship in relation to the oxidation of milk fat is discussed.