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Objective: Prior exposure to endotoxins such as lipopolysaccharide (LPS) may result in a transient state of immune refractoriness to subsequent challenge known as tolerance. Tolerance is thought to be a protective mechanism limiting excessive inflammation and organ injury, but it may impair the host’s ability to combat ensuing bacterial infection. Little data exists examining in vivo LPS tolerance specific to the lung environment. We investigated whether tolerance with intratracheal LPS prior to inducing Pseudomonas aeruginosa pneumonia will alter neutrophil recruitment, cytokine production, and the antimicrobial capacity of the murine lung. Design: Prospective, controlled animal trial. Setting: University research laboratory. Patients: Female ICR (CD-1) mice. Interventions: Mice were given 100ng LPS or normal saline by hypopharyngeal instillation under isoflurane anesthesia. Forty eight hours later, all mice received Pseudomonas aeruginosa, 5x10⁶ CFU, in 50uL HBSS by repeat intratracheal instillation. After four hours, bronchoalveolar lavage (BAL) fluid was collected to measure cytokine levels and cytospins were prepared for differential counting. Right lungs were harvested for myeloperoxidase assay and left lungs for bacterial culture. Main Outcome Measures: Cytokine levels, neutrophil recruitment, bacterial load. Results: TNF-alpha levels in BAL were significantly decreased in tolerant mice vs. nontolerant mice (1.1ng/mL+/-0.2 vs. 2.1+/-0.3, p=0.018). LPS also locally downregulated BAL neutrophil chemoattractants CXCL1 (p=0.045) and CXCL2 (p=0.006) in tolerant mice without affecting systemic levels. No reduction was seen in BAL neutrophil numbers or myeloperoxidase activity. Tolerant mice had at least a 2-fold increase in bacterial load at 24 hrs in BAL with 100 CFU/mL+/-38 vs. 43+/-17 and in lung homogenate with 100 CFU/mL+/- 35 vs. 39+/-15. Conclusions: While endotoxin tolerance with LPS significantly attenuates the host inflammatory cytokine response, it reduces bacterial clearance.