The ability of several Ly49 family members to inhibit natural killer (NK) cell functions through recruitment of SHP-1 phosphatase has been reported. In contrast, the mechanisms underlying the activating signal generated by Ly49D are poorly understood. A homodimeric phosphoprotein (pp16) that physically and functionally associates with Ly49D has been described. In this study, a rabbit anti-mouse pp16 antiserum was generated and used to demonstrate that pp16 corresponds to the recently described DAP12 molecule. In addition, we show that a second Ly49 family member that lacks an immunoreceptor tyrosine-based inhibitory motif and contains a charged residue in the transmembrane domain, Ly49H, also associates with DAP12. Furthermore, we show that engagement of the Ly49H/DAP12 complex results in phosphorylation of DAP12, intracellular calcium mobilization, and tumor necrosis factor secretion in transfected cells. These results thus provide evidence that Ly49H is an activating receptor that associates with DAP12, previously described as a pp16 component of the Ly49D receptor complex.