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Protease production by Bacillus subtilis with free and immobilized cells was examined in this study. Entrapment method of immobilization was used with gelatin, polyacrylamide, calcium alginate and agar matrices and protease production was studied compared to equivalent weight of free cells. Results showed that the gelatin entrapped cells produce 10.8 U/ml; the maximum enzyme titer followed by polyacrylamide, 9.22 U/ml; calcium alginate, 7.8 U/ml and agar, 6.48 U/ml. Equivalent free cells produce 5.7 U/ml protease enzyme. The cell leakage by gelatin was less, 0.06 mg/ml; by polyacrylamide, 0.22 mg/ml; calcium alginate, 0.32 mg/ml and the maximum, 0.74 mg/ml by agar. On repeated batch culturing it was observed that the gelatin entrapped cells can be reused for 7 cycles, followed by polyacrylamide, 6 cycles; calcium alginate, 5 cycles and agar for 3 cycles. The overall protease production by gelatin entrapped cells was 45.36 U/ml. The study suggests the gelatin, a better matrix than others for protease production by Bacillus subtilis.[New York Science Journal 2010; 3 (7): 20-24].(ISSN: 1554-0200).