Human erythrocyte acetylcholinesterase is an amphipathic enzyme whose hydrophobic membrane-binding domain can be selectively labeled with a lipophilic photoreagent and removed by digestion with papain. In this paper we demonstrate that methanolysis releases covalently bound fatty acids from the hydrophobic domain and thus confirm that this domain is a covalently linked glycolipid at the enzyme subunit C-terminus. About one mole of saturated and one mole of unsaturated fatty acids were released per mole of domain. Since the predominant unsaturated fatty acids (22:4 and 22:5) are minor components of the esterified fatty acid pool in human erythrocyte membranes, assembly of the glycolipid must involve a selected unsaturated fatty acid pool.