Molecule possessing ankyrin-repeats induced by lipopolysaccharide (MAIL) is a nuclear IkappaB protein recently identified as a molecule appearing in immunocompetent organs after administration of bacterial lipopolysaccharide (LPS). Participation of Toll-like receptor (TLR) 4, which is a major form of LPS receptors, in the LPS-induced MAIL expression was investigated. When a human myelomonocytic cell line U937 was treated with phorbol 12-myristate 13-acetate for 3 days, the LPS-induced MAIL expression was much potentiated in parallel with an increase in TLR4 expression. The MAIL induction was attenuated when the cells were treated with a neutralizing antibody against TLR4. The in vivo induction of MAIL in the spleen was smaller in mice having a missense mutation of the Tlr4 gene than in normal control mice. These results collectively indicate that TLR4 contributes, at least in part, MAIL induction after LPS stimulation.