The biological activity of deer antler has been considered to originate in the gangliosides, although the structures of gangliosides have not been well elucidated. The quality of deer antler as an Asian folk medicine has often been evaluated by the amount of gangliosides contained in the crude drug. We have completed the structural determination of five gangliosides isolated from deer antler in the present study. Five ganglioside fractions were isolated and purified from deer antler, Cervus nippon, by the Folch-Suzuki partition method, DEAE-Sephadex A-25, and further by silica gel column chromatography. High field proton nuclear magnetic resonance spectroscopy, gas chromatography/mass spectrometry, and fast atom bombardment-mass spectrometry studies characterized the isolated ganglioside fractions. GM3 and GD3 were present in the isolated ganglioside fractions. Samples were hydrolyzed in trifluoroacetic acid for direct compositional analysis and analyzed for sialic acid and neutral sugar without prior derivatization. Separation of the monosaccharides was achieved by HPLC on a Dionex CarboPac column eluted at a high pH. The resolved monosaccharides were identified using standard monosaccharides by pulsed amperometric detection. N-Acetyl GM3 (Neu5Ac), N-glycolyl GM3 (Neu5Gc), and N-acetyl GD3 (Neu5Ac) were present in the antler. The major ceramide moiety was composed of C16:0 or C22:0 fatty acids along with either C18 sphingosine or C20 eicosasphingosine.