Two local isolates designated as Bb 6 and Bb78 which characterized as Bacillus brev/'s and standard strain of ATCC 8185 were evaluated for their productivity of gramicidin S in G4/4 broth medium as well as their growth characteristics on Lauria broth medium. Production parameters for gramicidin S by the two isolates including incubation time, temperature, and pH were studied. Maximum production was found to be after 4 days and 6 days of incubation at 37 C and pH 7.2 for isolates Bb 6 and Bb 78 respectively. Nutritional requirements for production of gramicidin S were evaluated. Glycerol was found to be the preferred carbon source and stimulate production of gramicidin S when used at 10 mg / ml. However, fructose was found utilizable carbon source at that concentration. Mixture of different nitrogen sources (4.3 mg / ml ammonium oxalate, 1.5 mg / ml Proline, 1 mg / ml Phenylalanie, 1.3 mg / ml Histidine, 0.5 mg / ml Methionine, and 10 mg / ml Arginine) stimulate production optimally. However a concentrations of ammonium oxalate (4.3 mg / ml) and ammonium succinate (5 mg / ml) were also found to be good nitrogen sources. By using PC, TLC, and HPLC techniques, gramicidin S was characterized as the only product in the cultural medium of the two isolates. Factors for protoplast formation and regeneration were evaluated. Results indicated that treatment of cells with 6 mg / ml lysozyme for 20 minute at mid log phase convenient for obtaining a frequency for protoplast formation and regeneration of 99.3 %, and 31.71 % respectively. Treatments with 40 % polyethylene glyco! at room temperature for 2 minutes cause enhancement in frequency of protoplast fusion to 0.3. Forty recombinant cells were randomly selected and evaluated for produdtion of gramicidin S. variation in productivity was observed among tested recombinants. BbK4 was found giving the highest productivity (3650).