Medium improvement for the production of cholesterol oxidase (GO, EC 1.1.3.6) by Rhodococcus equi No. 23 was investigated using an orthogonal array design in two steps. Results revealed that yeast extract, Tween 80 and zinc sulphate had positive effects on CO production, but magnesium sulphate had an inhibitory effect. In addition, interaction between cholesterol and sodium chloride also had a significant effect on enzyme production. The improved medium consisted of 2.0 g/litre cholesterol, 8.0 g/litre yeast extract, 1.0 g/litre NH4Cl, 1.0 g/litre NaCl, 0.50 g/litre KH2PO4, 0.25 g/litre Na2HPO4, 0.10 g/litre L-valine, 0.15 g/litre L-tyrosine, 0.15 g/litre MgSO4.7H(2)O, 0.01 g/litre ZnSO4 . 7H(2)O, 0.10 g/litre FeSO4 . 7H(2)O and 4.0 ml/litre Tween SO. CO production at 60 h (about 0.24 units/ml) was about four-fold greater than with the control medium.