A novel O-acetylated GM3 containing 3-O-acetyl 4-sphingenine was isolated with one having a non-acetylated base from transplanted rat glioma tissue. The presence and position of the acetyl group were estimated by one- and two-dimensional proton nuclear magnetic resonance, and fast atom bombardment-mass spectrometries. In addition, the O-acetyl GM3 showed higher immunological activity toward anti-melanoma antibody in the presence of non-acetylated GM3 in complement-dependent liposome lysis than did non-acetylated or acetylated GM3 alone in the liposome, suggesting enhancement of immunological reactivity of the intact tumor cells by a small amount of O-acetyl GM3.