Toll-like receptor 4 (TLR4) is part of a group of evolutionarily conserved pattern recognition receptors involved in the activation of the immune system in response to various pathogens and in the innate defense against infection. We describe here the cloning and characterization of the avian orthologue of mammalian TLR4. Chicken TLR4 encodes a 843-amino-acid protein that contains a leucine-rich repeat extracellular domain, a short transmembrane domain typical of type I transmembrane proteins, and a Toll-interleukin-1R signaling domain characteristic of all TLR proteins. The chicken TLR4 protein shows 46% identity (64% similarity) to human TLR4 and 41% similarity to other TLR family members. Northern blot analysis reveals that TLR4 is expressed at approximately the same level in all tissues tested, including brain, thymus, kidney, intestine, muscle, liver, lung, bursa of Fabricius, heart, and spleen. The probe detected only one transcript of ca. 4.4 kb in length for all tissues except muscle where the size of TLR4 mRNA was ca. 9.6 kb. We have mapped TLR4 to microchromosome E41W17 in a region harboring the gene for tenascin C and known to be well conserved between the chicken and mammalian genomes. This region of the chicken genome was shown previously to harbor a Salmonella susceptibility locus. By using linkage analysis, TLR4 was shown to be linked to resistance to infection with Salmonella enterica serovar Typhimurium in chickens (likelihood ratio test of 10.2, P = 0.00138), suggesting a role of TLR4 in the host response of chickens to Salmonella infection.