In the course of a screening program for novel antibiotics showing antitumor activity, a cyclic peptide antibiotic sohbumycin was isolated from the culture broth of Streptomyces sp. No. 82-85, which was isolated from a soil sample collected in Kanagawa prefecture, Japan, and the preceding paper reported the isolation and physico-chemical properties of sohbumycin as well as the fermentation and taxonomy of the producing organism, Streptomyces sp. No. 82-85. Through careful fractionation of the fermentation broth from which sohbumycin was isolated, a new polyether antibiotic named octacyclomycin (1) was isolated, which showed both cytocidal activity against B16 melanoma cells and antimicrobial activity against Gram-positive bacteria in vitro, and showed no inhibitory activity against Gram-negative bacteria, yeast and fungi at the concentration of 500μg/ml. This paper deals with the isolation and structure elucidation of octacyclomycin (1). The 300-liter fermentation broth was mixed with 15 kg of Hyflo Super-Cel (Johns-Manville Co., U.S.A.) and then filtered with a filter press. The brown filtrate (260 liters) was adjusted to pH 6.0 and extracted with EtOAc (2 x 150 liters). The combined EtOAc layers were concentrated to approximately 10 liters, washed with H2O (5 liters), and dried over anhydrous Na2SO4. Concentration of the EtOAc layer resulted in a brown oil, which was chromatographed over silica gel. Fractions which showed antimicrobial activity against Micrococcus luteus were collected and further chromatographed over silica gel to afford octacyclomycin Na salt (51.7mg) as a colorless powder. The IR spectrum indicated the presence of hydroxyl (3460cm-1) and carboxylate (1589cm-1) functions in its structure. The 1H and 13C NMR spectral data summarized in Table 2 were obtained through 1H-13C two dimensional NMR spectrometry and 52 carbons and 83 hydrogens including 9 x CH3, 13 x CH2, 5 x CH, 3 x OCH3, 14 x OCH, 2 x C-O, 3 x O-C-O, 2 x O-CH-O and 1 x COO were observed. In the MS of octacyclomycin (1) Na salt, 1,039 ((M + 1)+) was observed. By the combination of the MS data and 1H and 13C NMR spectral data, molecular formula and molecular weight of octacyclomycin (1) Na salt and its free acid were concluded to be C52H87O19Na (MW 1,038) and C52H88O19 (MW 1,016), respectively. These physico-chemical and spectroscopical characteristics suggested that this compound was classified to be a polyether antibiotic. Because the established molecular formula (C52H88O19) of octacyclomycin (1) is different from these known antibiotics, 1 was shown to be a new polyether antibiotic.