Systematic examination by methods including GC-MS of the constituents in the benzene extract of Catalpa ovata cultured cells demonstrated the presence of menaquinone-1, dehydro-iso-α-lapachones, 1-hydroxy-2-methylanthraquinone etc. along with the known constituents of the wood of this plant such as catalpalactone and α-lapachones. Administration of 1-(^<14>C-carboxy)-o-succinylbenzoic acid (OSB) to the callus tissues followed by dilution analysis enabled us to confirm the incorporation of OSB into each of the callus constituents and to trap various biosynthetic intermediates. These results revealed the presence of the main biosynthetic pathway (COT→prenyl-COT→catalponone) and a subsidiary one (CHT→prenyl-CHT→catalponol) and also revealed the stereochemistry of prenylation. It was also clarified from the results of the administration of (1'-^3H, 1-(^<14>C-carboxy))-OSB to the intact Catalpa plant that OSB is directly cyclized to COT, which is then reduced to CHT.