Abstract 1. 1. Ascarosides and ascaroside esters in the total lipids of adult female Ascaris lumbricoides were fractionated by thin-layer chromatography. Ascarosides A, B and C were separated from phospholipids by development of thin-layer plates with polar solvents. Ascaroside esters appeared as seven distinct spots on chromatograms of neutral lipids. These compounds were found in all tissues, but in widely different amounts. Esters and free ascarosides were most abundant in the ovaries and uteri (fertilized eggs), respectively. The ascaroside layer of the egg shell consisted largely of free ascarosides. 2. 2. Acetic, propionic, α-methylbutyric and α-methylvaleric acids were present in ascaroside esters. Ascarosides contained only one glycone, whose chromatographic mobility was similar to that of ascarylose (3,6-dideoxy- L -arabinohexose). The aglycones were tentatively identified as a mixture of secondary alkanols containing C22,24,26,27,28,29,30,31,33,35,37 monols and C31,33,35,37 diols. C29 monols and C31,33 diols were most common. 3. 3. Each ascaroside and ascaroside ester fraction appearing on thin-layer chromatograms was a mixture, whose net chromatographic mobility was determined by chain length, number of free hydroxyl groups, number of glycosidic linkages and, in the case of ascaroside esters, by the nature of the volatile acid. 4. 4. Alkaline hydrolysis converted about 75 per cent of the chromatographic entity known as ascaroside A to ascaroside B. Evidence was obtained suggesting that naturally occurring ascaroside A contained a mixture of monol and diol aglycones in which one hydroxyl group of the diols was esterified with a higher fatty acid. The aglycones of ascarosides B and C were unesterified diols.