An extracellular cholesterol oxidase from Streptomyces fradine (PTCC 1121) was purified in one step using DEAE-Sepharose. The purified enzyme had a molecular weight of 60 KDa. The optimum pH and temperature for activity was found to be 7 and 70 degreesC, respectively. This cholesterol oxidase was stable in pHs between 4-10 at 4 degreesC until 4 h. Thermal stability experiments showed that it has high stability and retains its full activity at 50 degreesC for 90 min. K-m value for cholesterol oxidase was obtained to be about 7.06 X 10(-5) Mol. (C) 2001 Elsevier Science Inc. All rights reserved.