When S. fradiae was cultured in S medium, it stavted to produce neomycin in the middle of stationary phase of growth. Antibitoic production is regulated not only by glucose but also by metabolites formed from glucose. A chemically defined minimal salt broth was developen for the study of metabolites activating produition of antibiotic in a neomycin producer. When growth and production or antibiotic in minimal salt broth was examined with a full grown or a vefctativc mycelium, the medium was found not to be good for the growth, but to be good enough for the production of antibiotic with a full grown mycelium. When many carbotlydrates, organic acids, or alcohol were supplmented with instead of glucose in the medium suspcndcn with a full grown mycelium, the amount of antibiotic produced in the medium containing fumaratc was 5 times more than that in the medium with glucose. Further study indicated that the medium is not good also for the growth but good for the production of antibiotic. The antibiotic produced in this medium was identified to be neomycin. The activation of the production of neomycin by fumarate was further confirmed in a complex medium. Fuinarate is suspected to initiate and to activate the biosynthesis of neomycin at the gene level.