Technical Abstract: Arylamine N-acetyltransferases (NATs) are xenobiotic metabolizing enzymes well-characterized in several bacteria and higher eukaryotes. The role of NATs in fungal biology has only recently been investigated (Glenn and Bacon, 2009; Glenn et al., 2010). The NAT1 gene of Gibberella moniliformis was the first NAT cloned and characterized from fungi and is essential for the metabolism of antimicrobial compounds (benzoxazolinones) produced by cereals (maize, wheat, rye) and some wild grasses. We report a phylogenetic analysis employing an exhaustive annotated dataset of homologous NAT amino acid sequences recovered through inspection of 146 fungal genomes. We proceeded to amplification, exon-intron characterization and cloning of 16 NAT loci (3 of which are pseudogenes), predicted in Gibberella moniliformis, Gibberella zeae, Fusarium oxysporum, Aspergillus flavus and Aspergillus nidulans. Expression of recombinant NATs from these loci has enabled enzymatic profiling of their co-factor and substrate selectivity, as well as protein purification for future structure-function investigations. Real-time PCR quantification of the corresponding NAT transcripts is also in progress, supporting an effect of xenobiotics (including benzoxazolinone) to endogenous expression of certain NAT genes. Our findings suggest an association between fungal NAT metabolic capacity and affinity for hosts that produce benzoxazolinone defense compounds. Future studies will investigate more closely this possible association and its implications on agricultural management practices.