We investigated a possible coordination between the biosyntheses of two polyketides in the cereal head blight fungus Gibberella zeae, zearalenone (ZEA) and aurofusarin (AUR), which are catalyzed by the polyketide synthases (PKS) PKS4/PKS13 and PKS12, respectively. To determine if the production of one polyketide influences that of the other, we used four different transgenic strains of G zeae; three were deficient for either ZEA or AUR or both, and one was an AUR-overproducing strain. The mycelia of both the wild-type and ${Delta}PKS4$ strain deficient for ZEA produced AUR normally, whereas the mycelia of both the ${Delta}PKS12$ and ${Delta}PKS4::{Delta}PKS12$ strain showed no AUR accumulation. All the examined deletion strains caused necrotic spots on the surface of com kernels and were found to produce the nonpolyketide mycotoxins trichothecenes to the same amount as the wild-type strain. In contrast, the AUR-deficient ${Delta}PKS12$ strains produced greater quantities of ZEA and its derivatives than the wild-type progenitor on both a rice substrate and a liquid medium; the AUR-overproducing strain did not produce ZEA on either medium. Furthermore, the expression of both PKS4 and PKS13 was induced earlier in the ${Delta}PKS12$ strains than in the wild-type strain, and there was no difference in the transcription of PKS12 between the two strains. Therefore, these results indicate that the ZEA biosynthetic pathway is negatively regulated by the accumulation of another polyketide (AUR) in G zeae.