A comparative study of the glycolipids was performed on two cell lines of rat ascites hepatomas, island-forming and free cell types, and several marked differences were found as follows: 1. Hexose content in glycolipids derived from AH 7974F (free cell type) was about 3.6-fold as much as that from AH 7974 (island-forming cell type) on the basis of dry cell weight. 2. The glycolipids in the cells of AH 7974 were tentatively identified as glucosylceramide, lactosylceramide, galactosylgalactosylglucosylceramide, globoside and hematoside (GM3) by both thin-layer and gas-liquid chromatography. 3. On the other hand, the glycolipids in AH 7974F cells were glucosylceramide, lactosylceramide and at least four unknown lipids. 4. Structure of one of these unknown lipids was shown to be asialo-GM2 by methylation and enzymatic degradation studies. Moreover, the presence of asialo-GM1 was suggested by immunoprecipitation test with anti-asialo-GM1 serum. 5. Glucosylceramide and lactosylceramide from AH 7974F cells were found to possess hydroxy fatty acids as major fatty acids components, which were rare in these glycolipids from AH 7974 cells.