The mammalian Toll-like receptors (TLRs) activate cells of the innate immune system when stimulated by diverse ligands of microbial origin. In some instances, these ligands are directly engaged by the TLRs; however, this is not necessarily true in all cases. TLR2 recognizes multiple, structurally disparate microbial ligands, consistent with a requirement for co-receptors in ligand binding. Using N-ethyl-N-nitrosourea, we generated the recessive immunodeficiency phenotype oblivious, in which macrophages show diminished awareness of the S-enantiomer of the di-acylated bacterial lipopeptide MALP-2 and lipoteichoic acid, together with spontaneous ocular colonization by Gram-positive organisms and hypersusceptibility to Staphylococcus aureus infection. Oblivious macrophages readily detect the tri-acylated bacterial lipopeptide PAM3CSK4 as well as zymosan, revealing that some TLR2 ligands are activated via an Oblivious-independent pathway. The gene responsible for the oblivious phenotype has been positionally cloned. In its ability to carry the lipoteichoic acid and MALP-2 signal to the transmembrane signaling receptors TLR2 and TLR6, Oblivious serves a function analogous to CD14, which concentrates the lipopolysacchardide signal for transduction by TLR4. Besides microbial molecules, oblivious also serves as a receptor for endogenous molecules and may mediate (some) of the inflammatory events involved in the development of atherosclerosis.