A defined medium was developed in which to monitor deoxynivalenol (DON) accumulation, fungal growth and expression of genes involved in trichothecene biosynthesis (designated Tri genes). In liquid culture, DON accumulated shortly after maximum expression of Tri6 and coincident with expression of Tri5. This was generally 96 h after inoculation. The effects of sublethal concentrations of the fungicides azoxystrobin, trifloxystrobin, kresoxim-methyl and tebuconazole on biosynthesis of the trichothecene DON by Fusarium culmorum were studied using this medium. The strobilurin fungicides trifloxystrobin and azoxystrobin significantly reduced the accumulation of DON in culture medium at a range of concentrations. Kresoxim-methyl, also a strobilurin, and tebuconazole, a triazole, did not significantly reduce the accumulation of DON, although levels were lower than those in nonamended cultures. Trifloxystrobin significantly reduced the accumulation of DON when added to cultures before initiation of trichothecene biosynthesis. RT-PCR assays of the expression of Tri6 and Tri5 genes indicated that trifloxystrobin acted by inhibiting the initiation of trichothecene biosynthesis.