The effects of cobalt chloride on the production of trichothecene and ergosterol in Fusarium graminearum were examined. Incorporation experiments with (13)C-labeled acetate and leucine confirmed that both 3-acetyldeoxynivalenol and ergosterol were biosynthesized via a mevalonate pathway by the fungus, although hydroxymethyl-glutaryl CoA (HMG-CoA) from intact leucine was able to be partially used for ergosterol production. Addition of cobalt chloride at concentrations of 3-30 μM into liquid culture strongly enhanced 3-acetyldeoxynivalenol production by the fungus, whereas the amount of ergosterol and the mycelial weight of the fungus did not change. The mRNA levels of genes encoding trichothecene biosynthetic proteins (TRI4 and TRI6), ergosterol biosynthetic enzymes (ERG3 and ERG25), and enzymes involved in the mevalonate pathway (HMG-CoA synthase (HMGS) and HMG-CoA reductase (HMGR)) were all strongly up-regulated in the presence of cobalt chloride. Precocene II, a specific trichothecene production inhibitor, suppressed the effects of cobalt chloride on Tri4, Tri6, HMGS, and HMGR, but did not affect erg3 and erg25. These results indicate that cobalt chloride is useful for investigating regulatory mechanisms of trichothecene and ergosterol production in F. graminearum.