Itaconate biosynthesis was studied in intact cells of high-yield (RC4') and low-yield (CM85J) strains of the fungus Aspergillus terreus by methods (tracers, nuclear magnetic resonance spectroscopy, and mass spectroscopy) that did not interfere with metabolism. Itaconate formation in RC4' required de novo protein biosynthesis. Krebs cycle intermediates increased in both strains during the production of itaconic acid. The Embden-Meyerhof-Parnas pathway and the Krebs cycle were shown to be involved in this biosynthesis by using 14C- and 13C-labelled substrates and nuclear magnetic resonance spectroscopy. A metabolic pathway for itaconate formation from glucose in A. terreus is proposed.