In the fungus Fusarium sporotrichioides, biosynthesis of trichothecene mycotoxins requires at least three genetic loci: a core 12-gene cluster, a smaller two-gene cluster, and a single-gene locus. Here, we describe the Tri15 gene, which represents a fourth locus involved in trichothecene biosynthesis. Tri15 is predicted to encode a Cys(2)-His(2 )zinc finger protein and is expressed in a manner similar to genes in the core trichothecene gene cluster. However, disruption of F. sporotrichioides Tri15 does not affect production of T-2 toxin, the major trichothecene produced by this fungus. This result suggests that Tri15 is not necessary for the production of toxin. Cultures with exogenously added T-2 toxin have high levels of Tri15 expression and no detectable expression of the trichothecene biosynthetic genes Tri5 and Tri6. The expression analysis is consistent with Tri15 being a negative regulator of at least some of the trichothecene biosynthetic genes. In F. graminearum, Tri15 has been mapped to linkage group 2 and is therefore unlinked to the main trichothecene biosynthetic gene cluster.