Poly(gamma-glutamic acid) (PGA) production in Bacillus subtilis IFO3335 was studied. PGA was only slightly produced from medium (100 mi) containing 2 g citric acid and 0.5 g ammonium sulfate in B. subtilis IFO3335. When 0.01 g/100 ml L-glutamine was added to this medium, a large amount of PGA (0.45 g/100 ml), without any by-products such as polysaccharides, was produced. The changes in cell growth, and PGA, glutamic acid, citric acid and ammonium sulfate concentrations in this medium during cultivation were investigated. It was found that PGA was effectively produced for the short time of 20 h after an induction period and that glutamic acid was scarcely excreted during PGA production. PGA could be effectively produced using this medium containing L-glutamine, citric acid and ammonium sulfate. It is suggested that a small amount of L-glutamine added to the medium activated enzymes in the pathway of PGA synthesis in B. subtilis IFO3335. It can be presumed that the enzyme catalyzing the reaction from 2-oxoglutaric acid to L-glutamic acid was glutamate synthase in this bacterium.