Fusarium graminearum Z-3639 and F. sporotrichioides NRRL3299 produce the trichothecene mycotoxins 15-acetyldeoxynivalenol and T-2 toxin, respectively. These toxins differ in oxygenation at C-4, C-7, and C-8. In F. sporotrichioides, Tri1 (FsTri1) controls C-8 hydroxylation. To determine the function of an apparent F. graminearum Tri1 (FgTri1) homolog, both FsTri1 and FgTri1 genes were heterologously expressed in the trichothecene-nonproducing species F. verticillioides by fusing the Tri1 coding regions to the promoter of the fumonisin biosynthetic gene FUM8. FsTri1 and FgTri1 have been partially characterized by disruption analysis, and the results from these analyses suggest that FsTri1 most likely has a single function but that FgTri1 may have two functions. Transgenic F. verticillioides carrying the FsTri1 (FvF8FsTri1) converted exogenous isotrichodermin and calonectrin to 8-hydroxyisotrichodermin and 8-hydroxycalonectrin, respectively. Transgenic F. verticillioides carrying FgTri1 (FvF8FgTri1) converted isotrichodermin to a mixture of 7-hydroxyisotrichodermin and 8-hydroxyisotrichodermin but converted calonectrin to a mixture of 7-hydroxycalonectrin, 8-hydroxycalonectrin, and 3,15-diacetyldeoxynivalenol. A fourth compound, 7,8-dihydroxycalonectrin, was identified in large-scale F. verticillioides FvF8FgTri1 cultures fed isotrichodermin. Our results indicate that FgTri1 controls both C-7 and C-8 hydroxylation but that FsTri1 controls only C-8 hydroxylation. Our studies also demonstrate that F. verticillioides can metabolize some trichothecenes by adding an acetyl group to C-3 or by removing acetyl groups from C-4 or C-15. In addition, wild-type F. verticillioides can convert 7,8-dihydroxycalonectrin to 3,15-diacetyldeoxynivalenol.