BACKGROUND The authors compared the lung injury in rabbits that occurred after tracheal instillation of human breast milk (HBM) acidified to pH 1.8 with hydrochloric acid (HCl), HBM at its native pH (7.0), and HBM acidified with gastric juice to pH 1.8 and 3.0. METHODS The alveolar-to-arterial oxygen tension gradient and dynamic compliance were recorded before and hourly for 4 h after intratracheal instillation of 0.8 ml/kg HBM acidified with HCI (pH 1.8), HBM at its native pH (7.0), HBM acidified with gastric juice (pH 1.8 or 3.0), or 5% dextrose solution acidified with gastric juice (pH 1.8) as a control in 30 adult rabbits. The circulating neutrophil count and phagocyte oxidant activity were determined before and 1 and 4 h after instillation. RESULTS The alveolar-to-arterial oxygen tension gradient increased and dynamic compliance decreased significantly in all groups after instillation of HBM compared with baseline values and those in the control group. The severity of the lung injury after instillation of HBM at all pH values (1.8, 3.0, and 7.0) and after acidification with gastric juice or HCl was similar. The circulating neutrophil count increased steadily for 4 h after instillation (P < 0.013), whereas spontaneous phagocyte oxidant burst activity peaked at 1 h (P < 0.007) and returned to baseline by 4 h after instillation. CONCLUSIONS The severity of the lung injury after tracheal instillation of 0.8 ml/kg HBM in rabbits is similar at pH values between 1.8 and 7.0 after acidification with HCl or gastric juice. Tracheal instillation of HBM increases the circulating neutrophil count and phagocyte oxidant burst activity.