A mesophilic Bacillus sp. initially isolated from tiger excreta and later identified as a Bacillus subtilis strain was used to produce an extracellular cholesterol oxidase (C-OX) in cholesterol-enriched broth. This bacterial isolate was studied for the production of C-OX by manipulation of various physicochemical parameters. The extracellular C-OX was successfully purified from the cell-free culture broth of B. subtilis by successive salting out with ammonium sulfate, dialysis, and riboflavin-affinity chromatography. The purified C-OX was characterized for its molecular mass/structure and stability. The enzyme possessed some interesting properties such as high native Mr (105 kDa), multimeric (pentamer of similar to 21 kDa protein) nature, organic solvent compatibility, and a half-life of similar to 2 h at 37 A degrees C. The bacterial C-OX exhibited similar to 22 % higher activity in potassium phosphate buffer (pH 7.5) in the presence of a nonionic detergent Triton X-100 at 0.05 % (v/v). The K (m) and V (max) value of C-OX of B. subtilis C-OX were found to be 3.25 mM and 2.17 mu mol min ml(-1), respectively. The purified C-OX showed very little cytotoxicity associated with it.