Abstract Proteolipids from adult rat brain subcellular fractions were purified by a one-step procedure involving chromatography through Sephadex LH-60 eluted with an acidified chloroform—methanol mixture. The protein peak was eluted with the void volume and was free of adventitious lipids. The degree of purification was similar to that attained with the neutral—acidified chloroform—methanol dialysis method with the advantage that this new procedure can be carried out in only 3 h, with a recovery of proteins of 95–100%. Samples containing different lipid/protein ratios passed through the gel gave similar elution profiles. When labeled amino acids or palmitic acid were added to myelin total lipid extracts, no radioactivity was eluted with the protein, indicating that the proteolipid apoproteins purified by this method do not adsorb hydrophobic low-molecular-weight compounds.