The ruminal fate of the fat-soluble vitamins D and E was studied in dairy cows. Ten to 15 kg of ruminal contents was taken from each cow through a ruminal fistula. A sample was taken out (0-h sample) and the rest of the contents were mixed with 4,360 mg of all-rac-α-tocopheryl acetate (vitamin E; study 1) or 4,360 mg of all-rac-α-tocopheryl acetate, 250 mg of ergocalciferol (vitamin D(2)), and 250 mg of cholecalciferol (vitamin D(3); study 2). After mixing, the ruminal contents were returned to the respective cows. Blood was collected 0, 6, 24, and 30 h after introducing the vitamins into the rumen. Samples of ruminal contents were collected at 0, 1, 2, 4, 6, 24, and 30 h (in vivo). From the 1-h sample, 6 subsamples from each cow were incubated at 37 °C and taken out at 2, 4, 6, 12, 24, and 30 h (in vitro). In vivo concentrations of added α-tocopherol, ergocalciferol, and cholecalciferol in the rumen first increased and subsequently declined due to dilution effects of eating and passage out of the rumen. The level of the free-alcohol form of α-tocopherol from the natural content in feed was constant throughout the in vivo study, in contrast to the content of total α-tocopherol, which indicated that no hydrolysis of the acetate form into alcohol form happened in the rumen. In vitro, all added vitamins were found at constant levels; hence, none of the added vitamins were degraded in ruminal contents. The concentration of α-tocopherol in plasma increased at a rate per milligram of ruminally introduced α-tocopherol below the rate of the increase in plasma ergocalciferol or cholecalciferol metabolites per milligram of introduced ergocalciferol or cholecalciferol, respectively, over 24h. In conclusion, ergocalciferol, cholecalciferol, and α-tocopheryl acetate proved to be stable in the rumen and in ruminal contents from high-yielding dairy cows. Changes in plasma concentrations of the vitamins relative to the amount of vitamin introduced to the rumen indicated a lower effect on plasma status of ergocalciferol than of cholecalciferol, and an even lower effect of α-tocopherol. The limited plasma response after a single dose of α-tocopheryl acetate led to the conclusion that oral single dose therapy with all-rac-α-tocopheryl acetate is of limited physiological value.