Cytosporacin (1), a novel antibacterial polyketide containing naphthopyranone and isochromandione moieties, was isolated from the fermentation broth of the fungus Cytospora rhizophorae. A (1)H-detected ACCORD-ADEQUATE pulse sequence that distinguished (2)J(CH) from (3)J(CH) correlations provided critical information for structural determination. NOE studies established the relative configuration and revealed the presence of two rotamers. A biosynthetic (13)C-labeling experiment indicated that cytosporacin was derived from acetate origin.