Unidentified constituents in fresh pasture increase milk fat cis-9, trans-11 conjugated linoleic acid (CLA) concentration, and prevent milk fat depression, even though ruminal conditions conducive to reducing milk fat synthesis exist. One possible explanation is vitamin E (α-tocopherol), a constituent high in fresh pasture, but naturally low in conserved/dried forages and cereal grains. Twenty late-lactating dairy cows previously consuming a total mixed ration (TMR) were randomly allocated to one of two dietary treatments for 21 d: TMR (control; n=10); and TMR plus an additional 10000 i.u. α-tocopherol/d (VIT E; n=10). These cows were simultaneously compared with 13 late-lactation dairy cows previously grazing fresh pasture (PAS) balanced for age, parity and genetic merit. Average daily α-tocopherol intakes were approximately 468, 10520 and 1590 i.u./cow for the control, VIT E and PAS treatments, respectively. Dietary α-tocopherol supplementation (VIT E v. control) slightly increased milk fat content by 0·23 percentage units, but did not significantly alter milk fatty acid composition. Plasma trans-11 18[ratio ]1 (VA) content tended to increase and trans-10 18[ratio ]1 levels numerically declined following α-tocopherol supplementation suggesting possible changes in rumen biohydrogenation products. In addition, increased α-tocopherol intake in TMR-fed cows decreased serum urea levels and tended to alter milk fat 15[ratio ]0 suggesting changes in rumen microbial populations. However, when compared with cows grazing pasture, TMR-fed cows supplemented with α-tocopherol, still produced milk with lower cis-9, trans-11 CLA and VA, and higher trans-10 18[ratio ]1 concentrations suggesting α-tocopherol is not a primary reason for milk fatty acid profile differences between pasture and TMR-fed cows. Therefore, additional unknown pasture constituents favour production of fatty acids originating from the cis-9, trans-11 instead of the trans-10, cis-12 CLA biohydrogenation pathways.