Endotoxin-tolerant mice have mutations in Toll-like receptor 4 (Tlr4). This study identifies mutations in Tlr4 as the cause of endotoxin tolerance in two inbred mouse strains, C3H/HeJ and C57BL/10ScCr. Both strains are homozygous for a mutant Lps allele (Lps^d/d), which confers hyporesponsiveness to LPS challenge. Genetic and physical mapping of the Lps locus narrowed it to a 0.9-cM interval spanning 1.7 Mb. Three transcription units were identified within this interval, including Tlr4. C3H/HeJ mice have a point mutation in Tlr4, resulting in a nonconservative substitution of a highly conserved proline by histidine at codon 712. C57BL/10ScCr mice exhibit a deletion of Tlr4. These mutations strongly support the hypothesis that altered Tlr4 function is responsible for endotoxin tolerance. Tlr4 is a strong candidate for the Lps mutation due to its chromosomal location, presence of two independent mutant alleles, and its role in innate immune recognition of pathogens. The study provides evidence that Tlr4 is a critical regulator of the innate host response to bacterial LPS. The findings highlight the importance of Tlr4 in innate immunity and its potential role in the development of therapies for Gram-negative bacterial infections.Endotoxin-tolerant mice have mutations in Toll-like receptor 4 (Tlr4). This study identifies mutations in Tlr4 as the cause of endotoxin tolerance in two inbred mouse strains, C3H/HeJ and C57BL/10ScCr. Both strains are homozygous for a mutant Lps allele (Lps^d/d), which confers hyporesponsiveness to LPS challenge. Genetic and physical mapping of the Lps locus narrowed it to a 0.9-cM interval spanning 1.7 Mb. Three transcription units were identified within this interval, including Tlr4. C3H/HeJ mice have a point mutation in Tlr4, resulting in a nonconservative substitution of a highly conserved proline by histidine at codon 712. C57BL/10ScCr mice exhibit a deletion of Tlr4. These mutations strongly support the hypothesis that altered Tlr4 function is responsible for endotoxin tolerance. Tlr4 is a strong candidate for the Lps mutation due to its chromosomal location, presence of two independent mutant alleles, and its role in innate immune recognition of pathogens. The study provides evidence that Tlr4 is a critical regulator of the innate host response to bacterial LPS. The findings highlight the importance of Tlr4 in innate immunity and its potential role in the development of therapies for Gram-negative bacterial infections.