The natural killer (NK) cell activation receptor Ly49H is required for resistance to murine cytomegalovirus (MCMV). We show here that NK cell proliferation and production of interferon-γ (IFN-γ) was not dependent on Ly49H expression during early MCMV infection. During a later phase of infection, however, Ly49H+ NK cells selectively proliferated and this expansion was blocked by anti-Ly49H administration. With vaccinia virus infection, neither the early nor late phase of NK cell proliferation was selective for Ly49H+ NK cells. These findings indicated that Ly49H+ NK cells were specifically activated by MCMV and that MCMV infection was characterized by nonspecific and specific phases of NK cell activation in vivo.