A novel ganglioside which binds cholera-toxin B-subunit was purified from bovine brain by an h.p.l.c. system using an Aquasil column subsequent to Q-Sepharose column chromatography. T.l.c./immunostaining showed that the isolated ganglioside had about 60% of the binding reactivity of the authentic ganglioside GM1 for cholera-toxin B-subunit. On h.p.l.c., this ganglioside migrated between ganglioside GD1a and GD1b, and was found to give positive reactions with ninhydrin and fluorescamine reagents which specifically react with amino groups. The presence of a free amino group was further confirmed by chemical re-N-acetylation. The N-acetylated product had an identical RF value on h.p.l.c. and similar reactivity with cholera-toxin B-subunit as the authentic GM1. H.p.t.l.c., t.l.c./immunostaining, negative-ion fast-atom-bombardment (f.a.b.)-m.s., and 1H-n.m.r. spectroscopy of the novel ganglioside unequivocally demonstrated that it has the basal structure of GM1 with de-N-acetylated neuraminic acid instead of N-acetylneuraminic acid. In the present study we report for the first time that a ganglioside derivative containing de-N-acetylated neuraminic acid, de-N-acetylated GM1, exists in natural brain tissues.