Cultures of Aspergillus candidus produce, in addition to chlorflavonin, a closely related metabolite that has been isolated and characterized as the dechloro analogue. Radioactivity from [14C]dechlorochlorflavonin was incorporated irreversibly into chlorflavonin. Cultures supplemented with D-[U-14C]glucose, L-[methyl-14C]methionine, L-[U-14C]phenylalanine, or L-[β-14C]-phenylalanine yielded the 14C-labeled metabolites; chemical degradation of chlorflavonin labeled by the last named precursor showed that the isotope had been incorporated specifically into position 2 of the flavonoid carbon skeleton. We conclude that chlorflavonin is synthesized de novo by A. candidus and is, therefore, the first proven fungal flavonoid.