In the course of our research for new antibiotics produced by actinomycetes, new antibiotics, nanaomycins A, B, C and D, effective against mycoplasma, fungi and Gram-positive bacteria were obtained from the cultured broth of a strain which had been isolated from a soil sample collected at Nanao-shi, Ishikawa prefecture and designated Streptomyces rosa var. notoensis. Evidence is put forward which describes the structure and stereochemistry of nanaomycins A, B, C and D, as I, V, IV and VI, respectively. In order to study biosynthesis and to determine the position of the hydroxyl group in the naphthoquinone moiety, a feeding experiment with [1-^<13>C] acetate was effectively carried out. Nanaomycins are synthesized from 8 acetate units via a "polyketide" in S. rosa var. notoensis. Since the carbon atom adjacent to the phenolic hydroxyl group was enriched with [1-^<13>C] acetate, the hydroxyl group is not at C-6 position but must be at C-9 position. The absolute configuration was determined by ORD comparisons. The results indicated that nanaomycin D is an enantiomer of kalafungin produced by S. tanashiensis. The production of each enantiomer by two different species belonging to the genus Streptomyces is of interest in the biosynthesis of a series of these antibiotics.