Two new antibiotics, neothramycins A and B have been isolated from culture broth of Streptomyces No. MC916-C4, a strain belonging to the group C strains of cycloheximide-producing Streptomyces. We conclude from these data that neothramycins A and B are interconvertible isomers and that they belong to the anthramycin group antibiotics possessing a benzodiazepine structure. They may be distinguished from anthramycin, dextrochrysin and sibiromycin by their UV spectra. UV spectra of tomaymycins and neothramycins are very similar, but they are different in their molecular formulae and other spectra. Neothramycins A and B have weak activities against some bacteria and fungi. A marked prolongation in the survival period of mice implanted with the mouse leukemia L-1210 cells has been observed after treatment with neothramycin A or B intraperitoneally. In the treatment with daily intraperitoneal doses of 25-100 mcg of neothramycin A or B per mouse for 10 days, more than 200% of prolongation in the survival period of mice inoculated with EHRLICH ascites carcinoma cells were observed. Neothramycins A and B also inhibited multiplications of YOSHIDA rat sarcoma cells and C3H cells transformed by SV40 in tissue cultures. Acute LD50 of neothramycin A or B in mice was 20-30 mg/kg by the intravenous injection and 20-30 mg/kg by the intraperitoneal injection.