Tetrahymena pyriformis NT-I cells were grown in the medium supplemented with 1-O-hexadecyl glycerol which is the precursor for alkyl ether-containing phospholipids; choline phosphoglyceride and 2-aminoethylphosphonolipid, and alterations in the plasma membrane and microsome lipid composition were examined. No incorporation of supplemented 1-O-hexadecyl glycerol was seen in ethanolamine phosphoglyceride. 2. The hexadecyl glycerol fed membranes contain more polyunsaturated fatty acids than do the native membranes. However, the level of oleic acid (C 18:1) drops strikingly in the phospholipids of plasma and microsome membranes. 3. The hexadecyl glycerol-feeding induced a remarkable alteration in the polar headgroup composition of plasma membrane, especially a large increase in 2-amino-ethylphosphonolipid with a compensatory decrease in ethanolamine phosphoglyceride of plasma membranes. 4. The fatty acyl chain composition of phospholipids, especially ethanolamine phosphoglyceride, of the hexadecyl glycerol-fed plasma membranes and microsomes was found to be significantly different from that of the native membranes. 5. These results may indicate that marked alterations in polar headgroup as well as fatty acyl chain composition of membranes induced by glyceryl ether-feeding would be required for maintaining proper membrane fluidity.