Reasons for the variation in erythrocyte lipid extraction were investigated. We found that there was an uneven distribution of lipids after filtration (up to 10% accumulated in the filtrates) or centrifugation (up to 10% in the top fraction) of several apparently monophasic extraction systems. The uneven distribution was more pronounced in the systems containing relatively more water, or, generally speaking, with more nonpolar solvents. Ill-defined procedures using either filtration or centrifugation, therefore, could be the cause for variations in lipid extraction. A way to prevent the uneven distribution has been developed and is discussed.