Ansamycin antibiotics of the naphthalenoid subgroup are characterised by a bicyclic aromatic or quinonoid nucleus spanned by a macrocyclic lactam and biosynthesised from 3-amino-5-hydroxybenzoic acid (AHB). The C-30 hydroxyl substituent in actamycin, corresponding to the 4-position of AHB, is believed to be introduced late in the biosynthetic process. To address the mode of biological introduction of oxygen functionality at this site, an 18O labelling study was undertaken. Streptomyces sp. E/784 was fermented under an atmosphere containing 18O2, and actamycin was isolated. ES-MS analysis showed sodiated and potassiated molecular ion clusters enriched in 18O, while 13C NMR spectroscopy (including gradient heteronuclear multiple bond correlation spectroscopy) and methylation of actamycin confirmed that the C-28 quinonoid carbonyl and C-30 hydroxyl oxygens of actamycin were both derived from 18O2. Mass spectra displaying intense metallated molecular ions carrying a single 18O atom indicated that separate molecules of oxygen were involved at each site, confirming independent oxidation steps. This evidence establishes that the C-30 hydroxyl oxygen function of actamycin is introduced by an oxidation process involving O2, and analogous hydroxyquinone functionality in related ansamycins such as diastovaricin I and the hydroxyrifamycins is probably similarly derived.