Gibberella zeae is a pathogen of small grain crops and produces trichothecene mycotoxins in infected host tissue. The role of trichothecenes in the virulence of G. zeae was previously investigated using trichothecene-non-producing mutants that were generated via transformation-mediated disruption of a gene (Tri5) that encodes the first enzyme in the trichothecene biosynthetic pathway. The mutants were less virulent on some hosts than the wild-type strain from which they were derived. Here, we used two approaches to determine whether the reduced virulence of mutants was due specifically to Tri5 disruption or to non-target effects caused by the transformation process. First, we generated a revertant from a Tri5 disruption mutant by allowing the mutant to pass through the sexual phase of its life cycle. In approximately 2% of the resulting progeny the disrupted Tri5 had reverted to wild-type; however, only one of three revertant progeny also regained the ability to produce trichothecenes. In the second approach, we complemented the Tri5 mutation in a disruption mutant by transforming the mutant with a plasmid carrying a functional copy of Tri5. In all transformants examined, the ability to produce trichothecenes was restored. The restoration of trichothecene production in the revertant progeny and in the complemented mutant was accompanied by restoration of wild-type or near wild-type levels of virulence on wheat seedlings (cultivar Wheaton). The results indicate that the reduced virulence of the mutants was caused by disruption of Tri5 rather than non-target effects resulting from the transformation process. The results also provide further evidence that trichothecenes contribute to the virulence of plant-pathogenic fungi.