Toll-like receptor (TLR) is a key component in launching innate immune response to microbial challenge. TLR4 and TLR2 are recognized as specific receptors for components of Gram-negative and Gram-positive bacteria, respectively. Horses are extremely sensitive to endotoxin-induced cardiopulmonary distress and mortality which causes significant economic losses. To date, there are no data on the expression of TLR4 and TLR2 in horse lungs. Therefore, we examined the expression of TLR4 and TLR2 in lungs from normal or Escherichia coli lipopolysaccharide (E. coli LPS; 50 ng/kg; iv) treated horses. We also studied the impact of the depletion of pulmonary intravascular macrophages (PIM) on TLR4 and TLR2 expression in normal or LPS-treated horses. RT-PCR showed TLR4 mRNA but not TLR2, in normal horse lungs. PIM depletion reduced TLR4 mRNA expression without affecting TLR2. The LPS treatment increased the expression of TLR4 and TLR2 mRNA in normal and PIM-depleted horses compared to normal saline-treated horses. Light and electron microscopic immunocytochemistry showed TLR4 protein in PIM, alveolar macrophages and septal endothelium in lungs from normal or LPS-treated horses. Immuno-gold electron microscopy showed TLR4 in PIM and dual-label immuno-electron microscopy co-localized TLR4 and LPS in the cytoplasm and nucleus of PIM of LPS-treated horses. The present manuscript is the first report on the expression of TLR4 and TLR2 in normal and LPS-treated horses and direct co-localization of TLR4 with LPS molecules in PIM. These data provide evidence that PIM are equipped with TLR4 to handle and rapidly respond to circulating endotoxins.