Macrophages recognize the presence of infection by using the Toll-like receptor (TLR) family of proteins that detect ligands on bacterial, viral, and fungal pathogens. We show that murine macrophages stimulated with pathogen products known to signal through TLRs express ligands for the NKG2D receptor, found on NK cells, activated CD8+ T cells and activated macrophages. TLR signaling, through the MyD88 adaptor, up-regulates transcription of the retinoic acid early inducible-1 (RAE-1) family of NKG2D ligands, but not H-60 or murine UL16-binding protein-like transcript-1. RAE-1 proteins are found on the surface of activated, but not resting, macrophages and can be detected by NKG2D on NK cells resulting in down-regulation of this receptor both in vitro and in vivo. RAE-1-NKG2D interactions provide a mechanism by which NK cells and infected macrophages communicate directly during an innate immune response to infection.