Medium improvement for the production of cholesterol oxidase (COD) from Brevibacterium sp. employing response surface methodology (RSM) was investigated in this paper. Cholesterol dispersion improving factors, including surfactant Tween-80, solvent isopropanol and olive oil had a marked positive effect on COD production. The concentration of Tween-80, olive oil and cholesterol were further optimized by RSM. Results from RSM showed that the initial concentration of cholesterol and olive oil exerted a significant effect on COD production. These factors did not exert a significant effect on cell growth. The improved medium consisted of. 3.371 g/l cholesterol, 0.2125% (v/v) Tween-80, 0.2981% (v/v) olive oil, and COD production reached 1.285 U/ml after 36 h culture, 102% greater than the control medium. (C) 2002 Elsevier Science Ltd. All rights reserved.