Genetic evidence indicating that TOLL-like receptor 4 (Tlr4) is the lipopolysaccharide (LPS) receptor in mice was reported. However, biochemical evidence that murine Tlr4 confers LPS responsiveness has not been convincingly demonstrated. Inducible cyclooxygenase (COX-2) is selectively expressed in LPS-stimulated macrophages in part mediated through the activation of NF kappa B. Thus, we determined whether murine Tlr4 confers LPS responsiveness as evaluated by the activation of NF kappa B and COX-2 expression. Transfection of a murine macrophage-like cell line (RAW264.7) with the constitutively active form (delta Tlr4) of Tlr4 is sufficient to activate NF kappa B and COX-2 expression. However, the truncated form (delta Tlr4(P712H)) of the missense mutant Tlr4(P712H) found in LPS-hyporesponsive mouse strain (C3H/HeJ) inhibits LPS-induced NF kappa B activation and COX-2 expression. The inability of delta Tlr4(P712H) to activate NF kappa B and induce COX-2 expression is rescued by a constitutively active adapter protein myeloid differentiation factor 88 (MyD88), which interacts directly with the cytoplasmic domain of Tlr proteins. Furthermore, MyD88 is co-immunoprecipitated with the wild-type delta Tlr4 but not with the delta Tlr4(P712H) mutant. Together, these results indicate that Tlr4 confers LPS responsiveness in RAW264.7 cells and suggest that hyporesponsiveness of C3H/HeJ mice to LPS is attributed to the disruption of Tlr4-mediated signaling pathways that results from the inability of the mutant Tlr4(P712H) to interact with MyD88.