An anti-staphylococcal antibiotic, ericamycin was found in the culture of Streptomyces varius n. sp. by a Meiji group in 1966. In the 30 years since the first isolation of this antibiotic, no attempt has been made to elucidate the structure. This paper presents the structure determination of ericamycin using MS, NMR and CD spectral studies. Ericamycin, isolated as deep red crystals, showed characteristic UV, FAB-MS (pos.) m/z 500 (M+H)+, FAB-HR-MS (pos.) m/z 500.1359 (M+H)+ (calcd for C28H22NO8, 500.1346) data. Extensive NMR analyses including HMBC, HMQC and NOESY experiments allowed assignment of 1H, 13C and 15N signals. CD curves in MeOH showed a negative Cotton effect at 262nm (Δε=-33.7) and a positive Cotton effect at 242nm (Δε=+27.2), indicating counterclockwise helicity of substituted anthraquinone and isoquinolone rings and suggesting 6S,7S-pseudodiaxial protons conformation. Finally, the absolute structure of ericamycin was elucidated to be (6S,7S)-6,7-dihydro-6,10,15,16-tetrahydroxy-7-methoxy-3,11-dimethyl-2H-2-azahexaphene-1,9,14-trione, closely resembling benanomicinone (the aglycon of benanomicin A). Ericamycin is a promising lead for studies on the structure-activity relationships of dihydrobenzo[a]naphthacenequinone compounds.