Several members of the Ly-49 receptor family inhibit NK cell-mediated lysis of targets expressing appropriate MHC class I molecules. Ly-49D and Ly-49H, two Ly-49 molecules that lack immunoreceptor tyrosine-based inhibitory motifs (ITIM) in their cytoplasmic domains, associate with mouse DAP12, a molecule that possesses an immunoreceptor tyrosine-based activation motif (ITAM). Cotransfection of either Ly-49D or Ly-49H with DAP12 induces surface expression of both Ly-49 and DAP12. The Ly-49/DAP12 complex was coimmunoprecipitated from the transfected cells, demonstrating a physical association of DAP12 with Ly-49D or Ly-49H in the plasma membrane. Stimulation of transfectants with Abs recognizing either Ly-49D or Ly-49H results in cellular activation, as assessed by induction of tyrosine phosphorylation of multiple cellular substrates.