The fat-soluble vitamins A, D2, D3, E and K1, retinyl acetate, retinyl palmitate, tocopherol acetate, ergosterol and 7-dehydrocholesterol in milk were separated on a packed reversed-phase fused-silica microcolumn, and determined with UV detection. Conventional liquid chromatographic equipment was adapted for such purposes, providing a highly efficient and sensitive analytical system. The vitamins could be separated in gradient mode with detection limits comprised between 0.02 ng/ml for retinol and 2 ng/ml for vitamin E. All vitamins were separated in less than 17 min. Recovery studies showed good results for all solutes (89-107%) and the intra-day coefficients of variations ranged from 2 to 8%. Application of the proposed method to the quantification of fat-soluble vitamins in milk is reported.