1 4 C-labelled l-phenylalanine administered to shikonin-producing cell cultures of Lithospermum erythrorhizon was incorporated rapidly into deoxyshikonin and then into fatty acid esters of shikonin. A gradual increase in the radioactivity of shikonin derivatives was accompanied by a decrease in that of deoxyshikonin. Furthermore, 1 4 C-labelled deoxyshikonin supplied to cell cultures was incorporated into shikonin derivatives, e.g. acetyl- and β-hydroxyisovaleryl-shikonin, although 1 4 C-labelled shikonin was hardly converted into shikonin derivatives. 1 4 C-labelled deoxyshikonin incubated with a cell-free extract was also converted into esterified shikonin derivatives including acetylshikonin. These data strongly suggested that shikonin derivatives are biosynthesized from deoxyshikonin by hydroxylation and esterification at the C-1 position of its side chain both in cultured cells and in cell-free extracts of L. erythrorhizon.