AbstractRapid and extensive growth ofBacillus brevisATCC 9999 was obtained in a complex medium containing yeast extract and peptone. Gramicidin S (GS) production in this medium reached 2.5 g/liter and 0.25 g/g dry cell weight. GS synthetase I production was also high in this complex medium. Chemically defined media were also developed for this strain. In a glycerol‐ammonium sulfate‐Tris‐salts medium, the culture grew about 40% as well (rate and extent) as in complex medium. Although GS production was low (0.23 g GS/liter), peak specific activity of GS synthetase I was as high as on complex medium. Nutritional experiments showed that growth was stimulated by glutamine, methionine, proline, arginine, and histidine. Addition of these amino acids almost doubled the rate and extent of growth and GS production on a volumetric basis. However the increase in GS was due merely to the increased cell density; GS synthetase I specific activity was in fact decreased by the supplement. Complex medium is better than defined medium for GS and GS synthetase production due to increased cell density and a slower rate of synthetase disappea