Root cultures ofSwertia chirata(Gentianaceae) were grown with supplements of [1-13 C]glucose, [U-13 C6]glucose or [carboxy-13 C]shikimic acid. 1,3,5,8-Tetrahydroxyxanthone was isolated and analysed by quantitative NMR analysis. The observed isotopomer distribution shows that 1,3,5,8-tetrahydroxyxanthone is biosynthesized via a polyketide-type pathway. The starter unit, 3-hydroxybenzoyl-CoA, is obtained from an early shikimate pathway intermediate. Phenylalanine, cinnamic acid and benzoic acid were ruled out as intermediates