A specific and sensitive method based on high-performance liquid chromatography to measure vitamin K1 in rat liver is described. The procedure consists of three chromatographic steps, a preliminary purification of lipid extracts on Sep-Pak silica columns, a preparative stage using adsorption HPLC, and a final reversed-phase HPLC stage in which vitamin K1 is resolved from remaining contaminants and quantified by reference to an internal standard (vitamin K1 2,3-epoxide). The "within-run" coefficient of variation of the assay for vitamin K1 in 12 livers was 11.1% (n = 24). The concentration of vitamin K1 in liver of male rats fed a control diet containing no alfalfa was 8.0 ng/g (wet weight of liver) while female rat liver gave a mean value of 7.3 ng/g (wet weight of liver). Liver from male rats fed chow containing alfalfa gave a mean value of 44 ng K1/g (wet weight of liver).