The formation of 3-acetyldeoxynivalenol (ADON) and other secondary metabolites ofFusarium culmorumin a stirred jar fermentor is described in relation to changes in concentrations of sugars, N, P, and O2, and in pH in the medium as well as changes in cellular parameters. The addition of sodium [1-13C]acetate to the fermentation demonstrated that the conditions used resulted in the biosynthesis of ADON instead of other primary and secondary metabolites such as culmorin, dihydroxycalonectrin, and sambucinol for all but the early stages of the fermentation. Enrichment studies also revealed that dihydroxycalonectrin was not an important intermediate of ADON. Biosynthesis of ADON was apparently induced by nitrogen limitation. At the end of the fermentation, ca. 710 mg L−1ADON was obtained.