Streptomyces tenjimariensis SS-939 produced istamycins in a medium containing starch as the carbon source and soy bean meal as the nitrogen source. Istamycin production decreased substantially when starch was substituted with mono- or di- saccharides such as glucose, glycerol and maltose. A marked decrease of istamycin production was also observed when a rapidly used nitrogen source such as yeast extract, peptone or casamino acid was employed instead of soy bean meal. Addition of palmitate at a concentration of 0.2% doubled istamycin production. Istamycins A and B were found to be as active as fortimicin A and sporaricin A against Gram-positive and Gram-negative bacteria including aminoglycoside-resistant strains.