Technical Abstract: Corn produces the cyclic hydroxamic acids DIMBOA and DIBOA, which naturally transform into the more stable benzoxazolinones MBOA and BOA, respectively. These compounds are implicated in allelopathic weed suppression, insect feeding deterrence, and microbial disease resistance. Fusarium verticillioides, the most common fungal pathogen associated with corn, has the physiological capacity to detoxify MBOA and BOA. Biotransformation of BOA is suggested to involve hydrolysis (encoded by the FDB1 locus) to produce 2-aminophenol (2-AP), which is subsequently acylated (encoded by the FDB2 locus) to produce N-(2-hydroxyphenyl)malonamic acid (HPMA). Fungal growth is inhibited on BOA-amended medium if either locus is mutated. An fdb2 mutant can produce low levels of an acetylated 2-AP branch metabolite, N-(2-hydroxyphenyl)acetamide (HPAA). Suppression subtractive hybridization was used to identify genes up-regulated in response to BOA, and two gene clusters were identified that functionally correspond to the FDB1 and FDB2 loci. Genes at both loci are being evaluated. At the FDB2 locus a putative N-acetyltransferase (NAT) was of particular interest due to the postulated role of FDB2 in acylation of 2-AP. This gene was subcloned from an identified cosmid that complemented an fdb2 mutant. The subcloned gene also complemented an fdb2 mutation. Deletion of the gene eliminated the ability of F. verticillioides to metabolize BOA, and the mutants did not grow. We therefore functionally associate FDB2 as the gene encoding this putative NAT activity. The branch metabolite HPAA was produced at low concentrations in 'fdb2 mutants suggesting acetylation of the intermediate 2-AP occurred independently from the putative N-acyltransferase activity of FDB2, which is proposed to involve malonylation of 2-AP. Thus, we have provided further evidence for the genetics and biochemical nature of benzoxazolinone biotransformation that may enhance the ecological fitness of F. verticillioides in the cornfield environment.