Tri13 encodes a cytochrome P450 monooxygenase that catalyzes C-4 hydroxylation of some intermediates in the biosynthesis of type A and type B trichothecenes. This work demonstrated that Tri13 (FsTri13) of Fusarium sporotrichioides, a typical type A trichothecene producer, can participate in the biosynthesis of type B trichothecenes. When FsTri13 was expressed in Fusarium graminearum that produces 3-acetyldeoxynivalenol (3-ADON) as an end product, 3-acetylnivalenol (3-ANIV) was newly detected in the liquid medium. Although 3-ANIV can easily be prepared enzymatically from nivalenol (NIV) by using recombinant trichothecene 3-O-acetyltransferase (TRI101), there has been no conclusive report on the production of 3-ANIV in cultures of Fusarium strains. Possible biosynthetic pathway for production of 3-ANIV was suggested with regard to the function of Tri8, which is responsible for deacetylation of the side-chain O-acetyls in 3-ADON biosynthesis.