Improved isolation and effective purification of the main polar lipid (main glycophospholipid, MPL) from Thermoplasma acidophilum DSM 1728/10217 was achieved by a three-step procedure including (i) extraction of total lipid from lyophilized cells and removal of hydrophilic contaminants, (ii) fractionation of the total lipid via diethylaminoethyl (DEAE) cellulose chromatography and (iii) purification via a silica gel column by means of medium pressure liquid chromatography (MPLC). Adequate lipid analysis was accomplished by high performance liquid chromatography and high performance thin layer chromatography. The glycolipid fraction was clearly separated from the phospholipid by DEAE cellulose column chromatography, which can also be achieved by silicagel thin layer chromatography in two dimensions with different solvents. The advantage of the DEAE cellulose column is a one-step de-contamination from lipophilic pigments. The final purification of the MPL by MPLC on silicagel by isocratic elution with chloroform/methanol/water 80:25:3 (v/v/v) or petrolether/2-propanol/water 80:70:8 (v/v/v) proved an effective method to obtain pure MPL in relatively large quantity from great amounts of cell mass within a short time. Isocratic elution allows detection of the MPL peak via refraction index. Separation of MPL can be achieved within one hour by elution with chloroform/methanol/water and within 100 min by elution with petrolether/2-propanol/water. Up to 150 mg of purified MPL are obtained in a single run on a column with a diameter of 2.5 cm. Our study shows that MPLC provides an effective method for the isolation of pure MPL from archaeal lipid mixtures. (C) 2013 Elsevier B.V. All rights reserved.