Commercially available cholesterol esterase, cholesterol oxidase and peroxidase have been co-immobilized onto alkylamine glass beads (pore diameter 55 nm) through glutaraldehyde coupling with a conjugation yield of 2.3 mg/g of support and 76% retention of initial activity. The co-immobilized enzyme system showed maximum activity at pH 7.0, when incubated at 37 degreesC for 12 min. A method was developed for total serum cholesterol determination employing co-immobilized enzymes. There was a linear relationship between A(520) and cholesteryl acetate concentration ranging from 5 mg to 50 mg/dl reaction mixture. The minimum detection limit of the method is 50 mg/dl. Within day and between day coefficient of variation were <1.0% and <6%, respectively. A good correlation (r = 0.83) was found between the total serum cholesterol obtained by the present method and commercial Enzo-kit method employing free enzymes. Among the various serum substances tested at their physiological concentrations; Testosterone, vit D and progesterone caused 59%, 41% and 39% inhibition, while NaCl, KCl, CUSO4, creatinine, NaHCO3, albumin and estrogen had practically no effect. (C) 2002 Elsevier Science B.V. All rights reserved.