Abstract: Production of the polypeptide antibiotic bacitracin by a newly isolated Bacillus licheniformis B5 strain, using a laboratory medium was optimized. A medium of the following conditions was obtained: 1.5% glycerol as carbon source, 0.05% glutamic acid as nitrogen source, 1.43% inorganic phosphate, 0.0025% magnesium sulfate, with an initial pH of 7.5. Culture was incubated at 37°C for 48 h. The bacitracin productivity was about 174.2 units mL-1. Bacitracin antibiotic was also produced using immobilized cells of the B5 isolate in different immobilization supports (7.5% polyacrylamide, 4% sodium alginate beads and 2% agar). It was found that the highest bacitracin production rate (192 units mL-1) was obtained when using 7.5% polyacrylamide gel as an immobilization material.