A new antitumor antibiotic named mazethramycin has been isolated from the culture broth of Streptomyces thioluteus ME561-L4. It was easily converted into its methyl or ethyl ether and anhydromazethramycin during extraction and purification. In this communication, the isolation, characterization and structural elucidation of mazethramycin and its derivatives are reported. The structure of mazethramycin has been confirmed to be an N-methyl derivative of anthramycin.