The fungal entomopathogen Metarhizium robertsii (formerly known as M. anisopliae var. anisopliae) is a prolific producer of secondary metabolites of which very little is known at the genetic level. To establish the genetic bases for the biosynthesis of the mutagenic compound NG- 391, we identified a 19,818 kb genomic region harboring the predicted hybrid polyketide synthase-nonribosomal peptide synthetase NGS1, plus five additional ORFs. NGS1 knockouts generated by Agrobacterium-mediated transformation failed to produce detectable levels of NG-391, indicating the involvement of this locus in its biosynthesis. NGS1 deletion mutants had no significant changes in virulence levels against larvae of Spodoptera exigua and in resistance to hydrogen peroxide-generated oxidative stress compared to the wild-type strain. All 6 ORFs were expressed in medium supporting production of NG-391, and NGS1 was expressed during the interaction with the S. exigua host. The use of an NGS1 promoter-GFP reporter fusion showed that during in vitro growth in still broth cultures, NGS1 expression is restricted to the early exponential phase and is affected by M. robertsii cell density.