Abstract A high-performance liquid chromatographic method for the determination of chrysotalunin (1,1′, 8,8′-tetrahydroxy-3,3′-dimethyl-7,7′-bianthraquinoyl), the main hydroxyanthraquinone pigment in soils, is proposed. This method is based on the acetylation of chrysotalunin, and separation and determination of its tetraacetate by liquid chromatography. The minimum detectable amount of chrysotalunin expressed as chrysotalunin acetate was 10 pg. Seventeen samples of different soil types in Japan containing various amounts of carbon were examined. Chrysotalunin amounts ranged from 0.5 ppm (Orthic Acrisols) to 27.7 ppm (Humic Andosols). In addition, for comparison, all samples were assayed using a colorimetric method. From the results of these analyses, the chromatographic method was found to be suitable for the determination of chrysotalunin in soils of various types.