Toll-like receptors (TLRs) constitute an ancient family of pattern recognition receptors for conserved microbial structures that allow rapid detection of invading pathogens, triggering immune responses. TLR4 binds lipopolysaccharides (LPS) being involved in the recognition of Gram-negative bacteria. Herein we describe the generation and characterisation of a monoclonal antibody, named 3H3, against porcine TLR4. Its specificity was confirmed by reactivity with TLR4 expressing CHO cell transfectants. On peripheral blood leukocytes TLR4 was preferentially expressed on myelomonocytic cells, with monocytes expressing higher levels than granulocytes. Staining of lung tissue sections showed that TLR4 is also expressed on epithelial cells lining the bronchial tract, a distribution consistent with a surveillance function of bacterial invasion.