A rapid, sensitive analytical procedure was developed to quantitate alpha-tocopherol in rat liver. Liver is homogenized in acetone, fractionated by reversed-phase high-performance liquid chromatography and alpha-tocopherol quantitated by monitoring the column effluent at 280 nm. A single analysis requires 18 min. The method is linear from 1.0 to greater than 30.0 micrograms alpha-tocopherol per g of liver (wet weight). The average relative standard deviation of the slope of the standard curve is 2.3% and the single-day coefficient of variation is 6.3%. The use of an acetone extraction, reversed-phase column chromatography and quantitation of alpha-tocopherol at 280 nm results in a sensitive and reproducible system for the determination of alpha-tocopherol concentrations in rat liver. Preliminary studies have shown the assay to be useful for investigation of the effects of age and diet on hepatic alpha-tocopherol concentration.