The pollutant ozone (O₃) induces lung hyperpermeability and inflammation in humans and animal models. Among inbred strains of mice, there is a 3-fold difference in total protein (a marker of permeability) recovered in bronchoalveolar lavage (BAL) fluid after a 72-h exposure to 0.3 ppm O₃. To determine the chromosomal locations of susceptibility genes, we performed a genome screen using recombinant inbred (RI) strains of mice derived from O₃-susceptible C57BL/6J (B6) and O₃-resistant C3H/HeJ (HeJ) progenitors. Each RI strain was phenotyped for O₃-induced hyperpermeability, and linkage was assessed for 558 markers using Map Manager QTb27. A significant quantitative trait locus (QTL) was identified on chromosome 4. The likelihood ratio χ² statistic (16.6) for the peak of the QTL was greater than the significance threshold (16.3) determined empirically by permutation test. This QTL contains a candidate gene, Toll-like receptor 4 (Tlr4 ), that recently has been implicated in innate immunity and endotoxin susceptibility. The amount of the total trait variance explained by the QTL at Tlr4, the gene with the highest likelihood ratio statistic in the QTL, was approximately 70%. To test the role of Tlr4 in O₃-induced hyperpermeability, BAL protein responses to O₃ were compared in C3H/HeOuJ (OuJ) and HeJ mice that differ only at a polymorphism in the coding region of Tlr4. Significantly greater protein concentrations (430 ± 35 μ g/ml) were found in OuJ mice compared with HeJ mice (258 ± 18 μ g/ml) after exposure to O₃. Furthermore, reverse transcriptase/polymerase chain reaction analysis demonstrated differential expression of Tlr4 message levels between HeJ and OuJ mice after O₃ exposure. Together, results indicate that a QTL on mouse chromosome 4 explains a significant portion of the genetic variance in O₃-induced hyperpermeability, and support a role for Tlr4 as a strong candidate susceptibility gene.