Fusarium head blight (FHB) of wheat (Triticum aestivum L.), caused by the fungus Fusarium graminearum, is a major concern worldwide. FHB grain is reduced in yield, may fail to germinate, and is often contaminated with deoxynivalenol, a trichothecene mycotoxin linked to a variety of animal diseases and feed refusals. Annual losses in the tens of millions of dollars due to FHB underscore the need to develop improved methods of disease control and prevention. Previous research has identified deoxynivalenol biosynthesis as a virulence factor on wheat. Recently, we found that the TRI14 gene of F. sporotrichioides, closely related to F. graminearum, was not required for synthesis of a related trichothecene, T-2 toxin. TRI14 does not share similarity with any previously described genes in the databases. In this study, we examined the role that F. graminearum TRI14 may play in both deoxynivalenol synthesis and in virulence on wheat. TRI14 deletion mutants synthesize deoxynivalenol on cracked maize kernel medium and exhibit wild-type colony morphology and growth rate on complex and minimal agar media. However, FHB assays on greenhouse-grown wheat indicate that FgDeltaTri14 mutants cause 50-80% less disease than wild type and do not produce a detectable quantity of deoxynivalenol on plants. We discuss a number of possible roles that TRI14 may play in the disease process.