A fully enzymatic method for determining total cholesterol in human serum is described. The method appears especially suitable for adaptation to discrete mechanical analyzers either of the single channel or the multi-channel type. The method, which uses cholesterol esterase (EC 3.1.1.13), cholesterol oxidase (EC 1.1.3.6) and peroxidase (EC 1.11.1.7) with 4-aminophenazone and phenol as substrates in the indicator reaction, adapted to the Greiner Selective Analyzer GSA-II. For this purpose the critical parameters of the reaction were intensively examined. The complete reagent is stable within the GSA II dispenser at 4.degree. C for at least 1 wk. By omitting cholesterol oxidase in the blank reagent a sample blank and a partial reagent blank are obtained. Within a range up to 10.4 mmol/l (4.0 g/l) the maximum color is developed within 6 min. The calibration factor was stable for 4 mo. The method allows absolute measurements. At concentrations between 2-4 mmol/l within-batch precision ranged from 0.5-1.4%. Precision from day to day for the same control sera amounted to 2.8, 2.0, 2.7 and 2.0% for a period of 3 mo. Examination of accuracy yielded satisfactory results. Ascorbic acid in the physiological range had no significant effect on the results. Catalase or novaminesulfone added in vitro did not interfere. Optical interferences by bilirubin, Hb or turbidity are compensated by a sample blank. A comparison of results with the enzymatic method of Roeschlau et al. yielded satisfactory agreement. The limits of detection of the present method can be lowered by a factor of 2.2 by replacing phenol by dihalogen phenols.