A number of lymphocyte surface proteins are anchored in the cell membrane by glycophosphatidyl inositol (known as GPI) linkages instead of hydrophobic protein domains. Treatment of mouse T lymphocytes with antibodies specific for two such proteins, Thy-1 and Ly-6, are known to induce proliferation. We have found that antibodies specific for Qa-2, a GPI-anchored class I histocompatibility antigen, can also activate mouse T cells. To determine whether the GPI-anchor is important for this pathway of cell activation, we produced transgenic mice expressing either normal GPI-anchored Qa-2, or Qa-2 molecules with a membrane-spanning protein domain derived from H-2. Our studies show that only lymphocytes from transgenic mice carrying GPI-anchored forms of Qa-2 can be activated in vitro by Qa-2-specific antibodies. We also show that transgenic mouse T cells expressing a GPI-anchored form of H-2Db can be activated by anti-H-2Db antibodies. These results strongly indicate that the GPI-anchor is critical for this pathway of T cell activation.