The Fusarium trichothecenes T-2 toxin and deoxynivalenol (DON) are potent inhibitors of eukaryotic protein synthesis and are a significant agricultural problem. Three coregulated loci are required for T-2 toxin synthesis by Fusarium sporotrichioides. The core-trichothecene gene cluster consists of 12 genes (Tri3-Tri14) while the second locus consists of a single gene (Tri101). The third locus was recently partially described and encodes 1-2 biosynthetic enzymes and a putative regulatory gene. Here, we describe a detailed characterization of this locus. Located adjacent to Tri1 is Tri16, which is required for esterification of the C-8 hydroxyl. A putative regulatory gene, also adjacent to Tri1, is not required for T-2 toxin synthesis. The genomic sequence of Fusarium graminearum (a DON producer) contains a putative functional Tri1 and a nonfunctional Tri16. The presence of the Tri16 pseudogene is consistent with the chemical structure of DON, which has a C-8 keto group rather than the C-8 ester of T-2 toxin.