Previously, H. AOKI et al. reported the isolation of nocardicin A from the fermentation broth of Nocardia uniformis subsp. tsuyamaensis ATCC 21806. During further investigations to detect minor metabolites in the broth, a new antibiotic named nocardicin B was found to be produced together with nocardicin A by the same microorganism. Nocardicin B was isolated using Diaion HP 20 column chromatography (in the presence of sodium chloride) and preparative liquid chromatography. The purified nocardicin B decomposed at 262-264℃ and had the empirical formula C23H24N4O9·1/3H2O (confirmed by elemental analysis and Karl Fischer method). It is soluble in alkaline solutions (aqueous sodium hydroxide, ammonia, pyridine) and dimethyl sulfoxide but insoluble in other organic solvents. Its monosodium salt exhibited [α]D21 -162° (c 1, water) and gave positive reactions with ninhydrin and FeCl3-K3Fe(CN)6 reagents. The UV absorption spectrum of nocardicin B in pH 8.0 phosphate buffer showed two maxima at 223nm (E1%1cm 507) and 271nm (E1%1cm 181), and its IR and NMR spectra were also recorded. The chemical structure of nocardicin B was determined to be stereoisomeric to nocardicin A at the oxime group. Antimicrobial activities of nocardicin B (tested by serial agar dilution streak method) are shown in Table 2. Up to 750 mg/kg (intravenously, intraperitoneally, subcutaneously) or 2000 mg/kg (orally) of nocardicin B administered to mice caused no obvious toxic effects.