A N-glycolyneuraminic acid containing trisialoganglioside was isolated from bovine brains ganglioside mixture using Q-Sepharose. Its chemical structure was characterized as IV3NeuAc, II3NeuAc-NeuGc, Gg4Cer by gas-liquid chromatography, a permethylation study, sialidase degradation, TLC/enzyme-immunostaining, fast atom bombardment-mass spectrometry, fluorometric HPLC and proton nuclear magnetic resonance spectroscopy. This was unique in the mixed sialic acid constituents. (formula; see text) This accounted for 0.78% of the gangliosides. The ceramide structure was almost identical with those of major bovine brain ganglioside, as mainly composed of 18:0 fatty acid (90.9%) and d20:0 sphingosine base.