Cholesterol oxidase (EC 1.1.3.6) was isolated and purified from Staphylococcus epidermidis. The enzyme was purified with 60% ammonium sulphate followed by DEAE-Cellulose and Sephadex-200. The specific activity was 62 U mg^-1 protein and the purification fold was 43.4. The optimum temperature was 40^ºC and the optimum pH was 8.0. The K_m for cholesterol was 0.33 mM and for CaCl₂ was 0.23mM. The enzyme was inhibited by phenyl mercuric acetate (PMA), N-acetylimidazole (NAI), diethylpyrocarbonate (DEPC) and phenyl glyoxal (PG) indicating the presence of sulphhydryl, tyrosyl, histidyl and arginyl groups. The enzyme was activated by Ca²⁺ at 1.0 and 5.0 mM. Also Mn²⁺, Mg²⁺ and K⁺ were activators. Ba²⁺ and Zn²⁺ were inhibitors at the higher concentrations, whereas Al³⁺  and Cu ²⁺ were inhibitors at lower and higher concentrations.