The tissue culture ofRubia cordifolia var.pratensis andR. akane were performed to enhance the biosynthesis of anthraquinone pigments under various conditions. The production of alizarin and purpurin in the callus was separately analysed and was quantitatively compared. The pigment biosynthesis was more active in the callus fromR. cordifolia var.pratensis than fromR. akane. The addition of α-ketoglutaric acid, a biosynthetic precursor of anthraquinones, enhanced the production of alizarin and purpurin remarkably.