Lipid extraction efficiency is variable when different methods are used on the same tissue. After a systematic comparison on erythrocytes, we found that this variability was due to several factors, such as the addition of alcohol and chloroform as a mixture, the use of repeated extractions or partition procedures after the extraction. Most important, however, was the uneven distribution of solvents and lipids in an apparently monophasic extraction system. This formation reduces lipid extraction and causes accumulation of lipids in the extract supernatant, which leads to an overestimation of the extracted lipid content. We recommend a one-step 18.75 volume of methanol-chloroform (1.5:1 v/v) method for lipid extraction from erythrocytes, with methanol added before chloroform. This method combines simplicity in the extraction procedure, complete extraction of cholesterol and total phospholipids, and reliability in the quantitation of the extracted lipids.