The biosynthesis of cladospirone bisepoxide (1) was investigated by feeding 13C-labeled acetate to growing cultures of the fungus Sphaeropsidales sp. (strain F-24'707). 13C NMR spectral analysis demonstrated the polyketide origin of both naphthalene units. The origin of two epoxide oxygens was confirmed as from air by cultivation of the strain in an 18O2-enriched atmosphere. The [18O]incorporation pattern into palmarumycin C12 (11), the putative precursor of 1 led to the hypothesis that the carbonyl oxygen of 1 is derived from water by exchange of an oxygen atom. Inhibition of the biosynthesis of 1 with tricyclazole, an inhibitor of the 1, 8-dihydroxynaphthalene (DHN) melanin biosynthesis, confirmed the connection of both biosynthetic pathways.