Determination of the serum or plasma levels of retinol (vitamin A), 25-hydroxyvitamin D, and alpha-tocopherol (vitamin E) are the most frequently used parameters to evaluate status of vitamin A, D, and E, and also to assess the gastrointestinal absorption of the vitamins. We present a simple and sensitive method for simultaneous determination of these vitamins in 0.5 ml human serum or plasma. The vitamins were extracted from serum by methanol/iso-propanol (80/20, v/v) and n-hexane. The n-hexane phase was evaporated and injected to a reversed-phase (C-18) high-performance liquid chromatography system. Elution was performed with methanol/water (85:15, v/v) for 25-hydroxyvitamin D and retinol, and after that by methanol/water (98:2, v/v) for alpha-tocopherol. The eluate was monitored by a UV detector at 265 nm for detection of the vitamins. Baseline separation was obtained for all vitamins, and the system also permitted separate determinations of the D2 and D3 forms of 25-hydroxyvitamin D. The limit of detection and interassay variation for determination in 0.5 ml serum were 6.0 nmol/L and 6.2% for 25-hydroxyvitamin D2, 6 nmol/l and 6.1% for 25-hydroxyvitamin D3, 0.035 mumol/L and 5.0% for retinol, and 1.2 mumol/l and 5.5% for alpha-tocopherol.