The rapid and simple determination of fatty vitamins (vitamins A and E) in serum by high-performance liquid chromatography with a column-switching technique was investigated. The dilution of serum with an aqueous solution containing surfactant and organic solvent and the use of an aqueous solution with organic solvent as a sample pretreatment were an effective means of improving the sample recovery. To prevent sample decomposition during storage, the addition of an antioxidant reagent into the diluent was required. Under the optimal conditions, the relative standard deviation (R.S.D.) values against the standard samples were 1.12% (16.7 I.U./dl), 0.25% (333 I.U./dl) for vitamin A, and 1.02% (0.43 microgram/ml), 0.45% (8.5 micrograms/ml) for vitamin E, respectively. The relative coefficients (r2) between the sample amounts in serum and the peak areas were 1.0000 in the range from 16.7 to 667 I.U./dl (for vitamin A) and 0.9998 from 0.434 to 17.46 micrograms/ml (for vitamin E). The recoveries of vitamins from spiked serums were ca. 100% (vitamin A) and ca. 86% (vitamin E), respectively. By the combination of an on-line deproteination column and diluting solution, the simple and rapid determination of fatty vitamins could be routinely achieved in 18-min intervals.