Gilvocarcin V (GV), an antitumor agent produced by Streptomyces griseoflavus Gö 3592 and various other streptomycetes, is the most important representative of the distinct family of benzo[d]naphtho[1,2-b]pyran-6-one aryl C-glycoside antibiotics, which show excellent antitumor activity and a remarkably low toxicity. The most intriguing step of its biosynthesis is an oxidative rearrangement cascade, in which the C-5/C-6 of an angucyclinone precursor bond is broken. Although this oxidative cleavage is essential for the formation of GV's unique chromophore and for GV's biological activity, and is likely to occur similarly in the biosyntheses of other angucyclinone-derived antibiotics, such as the kinamycins and the jadomycins, it is only poorly understood. Herein we report various experiments which shed light onto this intriguing oxidative cleavage reaction. These include incorporation studies with 18O-labeled precursors and the isolation and structure determination of novel intermediates of gilvocarcin biosynthesis accumulated by mutants, in which two genes encoding monooxygenases responsible for the C-C-bond cleavage of the gilvocarcin pathway, gilOI and gilOIV, were deleted through targeted PCR.