A gas-liquid chromatographic method is described for the measurement of total cholesterol in serum. The method has been found to be simple, specific and precise. The results have been compared with the results of the enzymatic method of Röschlau, P., Bernt, E. and Gruber, W. (1974) (Z. Klin. Chem. Klin. Biochem. 12, 403) and with the results of an Auto-Analyzer method based on the manual method of Huang, T.C., Chen, C.P., Wefler, V. and Raftery, A. (1961) (Anal. Chem. 33, 1405) and Ness, A.T., Pastewka, J.V. and Peacock, A.C. (1964) (Clin. Chim. Acta 10, 229). The results of the gas-liquid chromatographic and of the enzymatic method show a high degree of correlation. The results of the Auto-Analyzer method are about 0.75 mmol/1 higher than those of the other two methods. The conclusion is drawn that the gas-liquid chromatographic method should be given consideration as a reference method for the measurement of total cholesterol in serum. It is a viable alternative for the generally accepted colorimetric reference method of Abell, L.L., Levy, B.B., Brodie, B.B. and Kendall, F.E. (1952) (J. Biol. Chem. 195, 357).