Some nutritional factors affecting the production of cholesterol oxidase (COX) by Rhodococcus equi no. 23 were investigated. Cholesterol and yeast extract respectively were the best carbon source and nitrogen source for the COX production. The optimum concentration of cholesterol and yeast extract was found to be about 0.1% and 0.4-0.5% (w/v) respectively. In addition, NH4Cl, NaCl and Tween 80 also exhibited enhancing effects on COX production, being maximal at 0.1% (w/v), 0.2% (w/v) and 1.0% (v/v) respectively. Moreover, optimal enzyme production occurred in medium that had an initial pH of 7.0.