In the course of screening for new antibiotics produced by Micromonospora, we discovered a series of 16-membered macrolide antibiotics, designated as izenamicins, produced by strain YS-02930K isolated from a soil sample collected at Izena island, Okinawa Prefecture, Japan. The antibiotics were found to consist of at least seven components designated izenamicins A1, A2, A3, B1, B2, B3 and B4. From the results of structure determination, izenamicins A3, B2 and B3 are new natural products although they have been prepared previously by chemical transformations of tylosin. This paper deals with the taxonomy of the producing strain, fermentation and purification of izenamicins. Based on morphological and chemical characteristics, the strain was identified as a Micromonospora sp. YS-02930K and deposited in the Fermentation Research Institute, Agency of Industrial Science and Technology, Japan, with the accession No. FERM P-7961. A stock culture of the strain was inoculated into seed medium and incubated, then transferred to production medium for fermentation. The broth filtrate was extracted with EtOAc and further processed to afford a crude powder of izenamicins, which was chromatographed on silica gel to obtain seven components as white amorphous powders. From physico-chemical analysis and NMR studies, izenamicins A3, B2, B3 were found to be identical with authentic antibiotics synthesized from tylosin, while A1, A2, B1, B4 were identical with rosamicin, juvenimicin A4, juvenimicin B1 and juvenimicin B3, respectively. Among the izenamicin components, izenamicin B3 showed the highest antimicrobial activity against Gram-positive and Gram-negative bacteria. An approximate ED50 of 89 mg/kg was observed when izenamicin B3 was administered orally to fasting mice challenged with Staphylococcus aureus Smith, and the antibiotic injected intravenously into mice showed an approximate LD50 value of 248 mg/kg. To our knowledge, this is the first report that izenamicins A3, B2 and B3 are produced by a microorganism, and we consider that izenamicin B3 (4'-deoxy mycaminosyl tylonolide) is an important intermediate for the synthesis of clinically useful 16-membered macrolide antibiotics.