The production of cholesterol oxidase (CO; EC 1.1.3.6) by Rhodococcus equi No. 23 was carried out in a 5-1 jar fermenter. A central composite experimental design and response surface methodology were employed to derive a statistical model for the effects of the initial cholesterol concentration and cultivation temperature on CO production. An initial concentration of 0.918 g/l cholesterol and a temperature at 39.4 degrees C were found to be optimal for the final enzyme production in batch cultures. The maximum CO production, after 24-h cultivation, predicted from the model was 0.278 U/ml. In addition. kinetic analysis revealed that enzyme production could be classified as the mixed type of growth associated at the optimized conditions. Moreover, the repression or inhibition of 4-cholesten-3-one on enzyme synthesis could be reduced, and CO production increased up to 0.340 U/ml, when the cells were cultivated with a strategy of two-stage culturing temperature (at 37.4 degrees C during the first 8 h and then at 39.4 degrees C till the end of the fermentation) and with the initial cholesterol concentration of 0.918 g/l.