We describe here the development of sandwich enzyme-linked immunosorbent assay (sandwich ELISA) and immunohistochemical staining for porcine interleukin-18 (PoIL-18) and their application to detection of PoIL-18 in vivo. Ten anti-PoIL-18 monoclonal antibodies (mAb), all of which were reactive with recombinant PoIL-18 by Western blotting, were established. Four (2-C-4, 9-H-6, 11-H-5, and 12-C-12) of 10 neutralized the biologic activity of PoIL-18 to induce interferon-y (IFN-gamma) from porcine peripheral blood mononuclear cells (PBMC). Four (2-C-4, 5-F-6, 9-H-6, and 12-C-12) of 10 were shown to be useful in immunohistochemical staining and detected PoIL-18 in Kupffer cells and macrophages in hepatic focal necrosis and macrophages in interstitial pneumonia in piglets with experimental endotoxemia using formalin-fixed, paraffin-embedded sections. A sandwich ELISA was developed using mAb 7-G-8 as a capture antibody and biotinylated mAb 5-C-5 as a detection antibody. This ELISA detected PoIL-18 with a minimum detectable concentration of 20 pg/ml and did not show cross-reactivity against PoIL-1beta, IL-8, IL-12, and IFN-gamma or murine and human IL-18. Using this ELISA, PoIL-18 was detected in the plasma and the bronchoalveolar lavage fluid (BALF) of pigs experimentally infected with Actinobacillus pleuropneumoniae. The availability of this ELISA and immunohistochemical staining for PoIL-18 may contribute to a further understanding of the role of this cytokine in various porcine immune responses and diseases.