NK cells participate in innate immune responses by secreting gamma interferon (IFN-gamma) and by destroying virus-infected cells. Here the interaction between influenza A or Sendai virus-infected macrophages and NK cells has been studied. A rapid, cell-cell contact-dependent production of IFN-gamma from NK cells cultured with virus-infected macrophages was observed. Expression of the MHC class I-related chain B (MICB) gene, a ligand for NK cell-activating receptor NKG2D, was upregulated in virus-infected macrophages suggesting a role for MICB in the activation of the IFN-gamma gene in NK cells. IL12Rbeta2, IL18R and T-bet mRNA synthesis was enhanced in NK cells cultured with virus-infected macrophages. Upregulation of these genes was dependent on macrophage-derived IFN-alpha. In contrast to IL12Rbeta2, expression of WSX-1/TCCR, a receptor for IL27, was reduced in NK cells in response to virus-induced IFN-alpha. In conclusion, these results show that virus-infected macrophages activate NK cells via cytokines and direct cellular interactions and further emphasize the role of IFN-alpha in the activation of innate immunity.