Species of the genus Fusarium produce a great diversity of agriculturally important trichothecene toxins that differ from each other in their pattern of oxygenation and esterification. T-2 toxin, produced by Fusarium sporotrichioides, and nivalenol (NIV), produced by some strains of F. graminearum, contain an oxygen at the C-4 position. Deoxynivalenol (DON), produced by other strains of F. graminearum, lacks a C-4 oxygen. NIV and DON are identical except for this difference, whereas T-2 differs from these trichothecenes at three other carbon positions. Sequence and Northern analyses of the F. sporotrichioides genomic region upstream of the previously described core trichothecene gene cluster have extended the cluster by two genes: TRI13 and TRI14. TRI13 shares significant similarity with the cytochrome P-450 class of enzymes, but TRI14 does not share similarity with any previously characterized proteins. Gene disruption and fermentation studies in F. sporotrichioides indicate that TRI13 is required for the addition of the C-4 oxygen of T-2 toxin, but that TRI14 is not required for trichothecene biosynthesis. PCR and sequence analyses indicate that the TRI13 homolog is functional in NIV-producing strains of F. graminearum but nonfunctional in DON-producing strains of the fungus. These genetic observations are consistent with chemical observations that biosynthesis of T-2 toxin and NIV requires a C-4 hydroxylase while biosynthesis of DON does not.