Purpose of work: The biosynthetic pathway of a new antitumor compound, haloroquinone, is elucidated to facilitate metabolic regulation for product accumulation and modification to produce new bioactive structural analogues of the compound. The biosynthetic origin of a novel promising protein kinase B inhibitor and anti-tumor compound, haloroquinone, from a marine-derived fungus, Halorosellinia sp. was clarified. The origin of carbon skeleton of haloroquinone was elucidated by feeding experiments with [2-C-13] malonate and [1,2,3-C-13(3)] malonate followed by C-13-NMR analysis of the isolated compounds: 15 carbon atoms were derived from malonate, of which eight were from the methylene group and seven from the carboxyl group. The remaining one is probably obtained by O-methylation. Haloroquinone is thus synthesized via a polyketide pathway using malonyl-CoA as both the starter unit and the extender unit.