Cytotoxic treatment with rabbit antiserum raised against purified glycosphingolipid “asialo GM1” was capable of eliminating natural killer (NK) activity of spleen cells from different inbred mouse strains including CBA/J, C57BL/6, BALB/c, AKR, and athymic nude mice. The anti‐asialo GM 1 antiserum showed little cross‐reactivity with structurally related glycolipids, e.g. GM 1, GD 1 b and asialo GM 2 in the microflocculation test. The specific reactivity of this antiserum with NK cells was confirmed by the quantitative absorption of anti‐NK activity with graded amounts of asialo GM 1 but not with other glycosphingolipids. The absorption of anti‐brain‐associated T cell antigen (anti‐BAT) with asialo GM 1 also effectively diminished its anti‐NK activity, leaving the ability to kill T cells intact. This suggests that the antibody to asialo GM 1 is responsible for the anti‐NK activity contained in the anti‐BAT antiserum. In contrast to the extreme sensitivity of NK cells to anti‐asialo GM 1, alloreactive cytotoxic T killer cells generated in the mixed lymphocyte culture were not killed by anti‐asialo GM 1 and complement. These results indicate that asialo GM 1 is expressed on mouse NK cells in a high concentration.