Two chlorine-containing antibiotics, clazamycins A and B, were isolated from the culture broth of Streptomyces No. MF990-BF4 which is closely related to Streptomyces violaceorectus and Streptomyces cinereoruber. The strain was cultured, and the antibiotics were purified via column chromatographies on Amberlite IRC-50, Amberlite XAD-2, and activated carbon. Clazamycin A hydrochloride's structure was determined to be (5S,6aR)-5-chloro-4,5,6,6a-tetrahydro-6a-hydroxy-3-imino-3H-3a-azapentalene by X-ray crystallographic analysis, while clazamycin B was confirmed as its 6a-epimer through spectral data and equilibrium conversion studies. Their physicochemical properties (molecular formula C7H9N2OCI for both, melting point, specific rotation, UV, IR, PMR, CMR, MS), stability (pH-dependent interconversion between A and B), antibacterial activities (weak against various bacteria as shown in Table 3), and in vivo activities (prolonged survival of mice inoculated with leukemia L-1210 cells, intravenous LD50 of 50–100 mg/kg for A and >100 mg/kg for B) were investigated. Dihydroclazamycin A, prepared by hydrogenation, had no antibacterial activity.