Most Fusarium graminearum isolates produce low or undetectable levels of trichothecenes in liquid shake cultures, making it difficult to perform biochemical studies of trichothecene biosynthesis. To develop strains with higher levels of trichothecene production under liquid shake conditions we transformed F. graminearum with both a reporter gene containing a homologous trichothecene pathway gene promoter (TRI5) and a gene encoding a heterologous trichothecene pathway transcription factor (TRI6). The TRI5 and TRI6 genes are part of the trichothecene pathway gene clusters of both Fusarium sporotrichioides and F. graminearum. These genes encode trichodiene synthase (encoded by TRI5), the first enzyme in the trichothecene pathway, and a transcription factor (encoded by TRI6) required for pathway gene expression. Transformation of F. graminearum with plasmids containing either an F. graminearum TRI5 promoter fragment (FGTRI5^P) or FGTRI5^P coupled with the -D-glucuronidase (GUS) reporter gene resulted in the identification of several transformants capable of producing 45 to 200 mg of 15-acetyldeoxynivalenol (15-ADON)/liter in liquid shake culture after 7 days. Increased 15-ADON production was only observed in transformants where plasmid integration occurred through the FGTRI5^P sequence and was not accompanied by increased GUS expression. 15-ADON production was further increased in liquid culture up to 1,200 mg/liter following introduction of the F. sporotrichioides TRI6 gene (FSTRI16) into F. graminearum. The effects of FSTRI6 on 15-ADON production also depended on plasmid integration via homologous recombination of the FGTRI5^P fragment and resulted in a 100-fold increase in GUS expression. High-level production of 15-ADON in liquid shake cultures provides a convenient method for large-scale trichothecene preparation. The results suggest that targeting transformation vector integration to FGTRI5^P alters pathway gene expression and are consistent with the proposed conservation of TRI6 function between Fusarium species.