Several inhibitors of fatty acid synthesis have recently been noted as a new group of antibiotics which show selective toxicity against procaryotes. Thus, thiolactomycin1~3) selectively inhibits the fatty acid synthetase of Escherichia coli (type II) but has little effect on that of mammalian tissues (type I)4-8). On the other hand, cerulenin inhibits both type I and type II synthetases4,7). In order to find new inhibitors of the fatty acid synthetase of E. coli (type II), we employed a fatty acid synthetase assay system comprising fraction A and the acyl carrier protein (ACP), which was prepared by essentially the same method as described by MAJERUS et al.8) from E. coli K12 except that the ACP preparation was used without further purification by DEAE-cellulose and DEAE-Sephadex chromatography. The activity of the fatty acid synthetase was determined by the radioactive assay method described by KAWAGUCHI et al.9), which measured the incorporation of [2-14C]malonyl-Co A into the fatty acid fraction in the presence of acetyl-Co A and NADPH. Three active compounds were isolated from the culture filtrate of Streptomyces strain No. S-1998. These antibiotics belonging to the ansamycin group were named naphthoquinomycins A, B and C. The strain No. S-1998 was cultivated on a rotary shaker at 27°C for 5 days in 5-liter Erlenmeyer flasks containing 1 liter of a medium consisting of glycerol 3.0%, corn steep liquor 1.0%, dry yeast 0.3%, NaCl 0.5% and CaCO3 0.35%. The filtered fermentation broth (3 liters, pH 7.1) was adjusted to pH 3.0 with HCl and extracted with EtOAc. The organic layer was concentrated in vacuo and subjected to silica gel column chromatography. The active fraction eluted with CHCl3 - MeOH (100: 2) was evaporated in vacuo to give a crude material.