Overexpression of the Pseudomonas virulence factor ( pvf) biosynthetic operon led to the identification of a family of pyrazine N-oxides (PNOs), including a novel dihydropyrazine N,N'-dioxide (dPNO) metabolite. The nonribosomal peptide synthetase responsible for production of (d)PNOs was characterized, and a biosynthetic pathway for (d)PNOs was proposed. This work highlights the unique chemistry catalyzed by pvf-encoded enzymes and sets the stage for bioactivity studies of the metabolites produced by the virulence pathway.