Although activation of Toll-like receptor 4 (TLR4)-positive cells is essential for eliminating Gram-negative bacteria, overactivation of these cells by the TLR4 ligand LPS initiates a systemic inflammatory reaction and shock. Here we demonstrate that SPRET/Ei mice, derived from Mus spretus, exhibit a dominant resistance against LPS-induced lethality. This resistance is mediated by bone marrow-derived cells. Macrophages from these mice exhibit normal signaling and gene expression responses that depend on the myeloid differentiation factor 88 adaptor protein, but they are impaired in IFN-β production. The defect appears to be specific for IFN-β, although the SPRET/Ei IFN-β promoter is normal. In vivo IFN-β induction by LPS or influenza virus is very low in SPRET/Ei mice, but IFN-β-treatment restores the sensitivity to LPS, and IFN type 1 receptor-deficient mice are also resistant to LPS. Because of the defective induction of IFN-β, these mice are completely resistant to Listeria monocytogenes and highly sensitive to Leishmania major infection. Stimulation of SPRET/Ei macrophages leads to rapid down-regulation of IFN type 1 receptor mRNA expression, which is reflected in poor induction of IFN-β-dependent genes. This finding indicates that the resistance of SPRET/Ei mice to LPS is due to disruption of a positive-feedback loop that amplifies IFN-β production. In contrast to TLR4-deficient mice, SPRET/Ei mice resist both LPS and sepsis induced with Klebsiella pneumoniae.