Mechanical ventilation of preterm infants is associated with pulmonary inflammation. Intubated infants often develop bacterial tracheal colonization, but little is known about endotoxin in tracheal aspirates (TAs) or the mobilization of innate immunity toward endotoxin, a potent stimulus that contributes to inflammatory disease. We characterized mobilization of endotoxin-directed innate immunity in TAs from an observational cohort of mechanically ventilated neonates. TA supernatants (n = 42; GA = 23-40 wk, postnatal age = 1-71 d) were assayed for endotoxin (Limulus amoebocyte lysate assay) and endotoxin-modulating proteins: bactericidal/ permeability-increasing protein (BPI), LPS-binding protein (LBP), and soluble cell differentiation antigen 14 (sCD14). TA cellular BPI was measured by ELISA, Western blot, flow cytometry, and bactericidal assay. TA mRNAs encoding endotoxin-modulating proteins were measured by quantitative real-time PCR (qRT-PCR). Endotoxin in TA supernatants was proportional to both postnatal age and polymorphonuclear leukocytes (PMN). Neonatal TAs were rich in PMN containing BPI and expressed mRNAs encoding Toll-like receptor (TLR) 4, CD14, and myeloid differentiation protein 2 (MD-2). Extracellular BPI was consistently detectable and correlated with TA PMN and GA. Both extracellular- and cellular-BPI increased during the first postnatal week. TA extracellular BPI, LBP, and sCD14 were positively correlated. TAs from intubated neonates demonstrate endotoxin accumulation and mobilization of endotoxin-directed innate immunity, potentially contributing to pulmonary inflammation.