We previously reported a hamster animal model of melanoma in which the tumor tissue expresses gangliosides GM3, GD3, and O-acetyl GD3. This ganglioside pattern is similar to that in human melanomas (Ren, S., A. Slominski, and R. K. Yu. 1989 Cancer Res. 49: 7051). In this study, we isolated and purified these gangliosides using chloroform-methanol extraction, Folch partition, chromatographies on DEAE-Sephadex A-25, and Iatrobeads columns. The yields of gangliosides GM3, GD3, and O-acetyl GD3 were 44.1 mg, 19.6 mg, and 9 mg per 100 g of Ma melanotic melanoma tissues, respectively. The structures of these gangliosides were characterized by periodate oxidation, gas chromatographic (GC) analysis, fast-atom bombardment-mass spectrometry (FAB-MS), and nuclear magnetic resonance (NMR) studies. The structure of hamster melanoma O-acetyl GD3 is different from the 9-O-acetyl GD3 previously reported in human melanoma. The major fatty acids of this ganglioside are C16:0, C18:0, C20:0, C22:0, and C24:0 and the longchain base is C18-sphingosine.