Berninamycins A, a potent antibacterial agent which was originally isolated from Streptomyces bernensis. In this report, berninamycin A was isolated fromacetone extract of S. atroolivaceusNBRC12741 (type strain) and identified using NMR and MS spectra. The identification of berninamycin A was accomplished by analysis of NMR experiments including COSY, HMQC, HMBC, ROESYspectra. Since the absolute stereochemistry of berninamycinAwas not determined before, in this report the absolute stereochemistries of Thr and hydroxylVal in berninamycinAwere determined to be L-formby chemical analysis ofmodifiedMarfeyÂs Method. Taken together with analysis ROESY spectrum, the whole absolute chemistry of berninamycinAwas determined. The production of berninamycin A by S. atroolivaceus was estimated as high as 19 ïg/mg (quantity of berninamycin A /wet weight of whole cells and agar) by HPLC analysis, which indicated that S. atroolivaceuswas efficient producer of berninamycin A.