There are 4 P450 oxygenases involved in the biosynthesis of T-2 toxin in Fusarium sporotrichioides. Exactly how these enzymes react to antimicrobial plant defense compounds is unknown. Xanthotoxin (8-methoxypsoralen) is a phototoxic furanocoumarin that acts as a P450 oxygenase inhibitor. The current study shows that the addition of concentrations of 1.0 mmol/L or less of xanthotoxin to liquid cultures of F. sporotrichioides NRRL3299 can effectively block T-2 toxin production and cause an increase in accumulation of trichodiene, the hydrocarbon precursor of trichothecenes. The addition of xanthotoxin to liquid cultures of a trichodiene-accumulating F. sporotrichioides Tri4- mutant caused a 3- to 10-fold increase in trichodiene accumulation, suggesting that xanthotoxin not only blocks trichothecene oxygenation reactions, but may in some way also promote the synthesis of trichodiene. Feeding studies showed that 2 of the 4 P450 oxygenases, TRI4 and TRI1, were more sensitive to xanthotoxin, while oxygenases TRI11 and TRI13 were unaffected. Quantitative reverse-transcriptase PCR indicated that several of the genes in the toxin biosynthetic pathway were upregulated by xanthotoxin, with Tri4 showing the highest increase in expression. These results indicate that while xanthotoxin inhibits specific P450 oxygenase activity, it also has an effect on gene expression.