This study was carried out to enhance the production of sisomicin by Micromonospora inyoensis ATCC 276000. The effect of various sugars and organic acids as the supplements to a basal mineral medium was tested for the sisomicin production by fully grown mycelium. Among the substances tested, acetate and citrate greatly increased the production of sisomicin at the sixth day of culture. Especially, in the basal mineral medium containing 0.1M sodium acetate, sisomicin production was 6.7 times more than that in the same medium with glucose. When 0.1M sodium acetate was added in the fermentation medium initially, the cell growth was inhibited by sodium acetate, although specific productivity was higher than that in the same medium without sodium acetate. On the contrary, when sodium acetate was added to the culture after three days, the sisomicin production and specific productivity were 1.6 times higher than those in the same medium without sodium acetate. The results suggested that sodium acetate was a stimulating substance of sisomicin production by M. inyoensis.