We prospectively evaluated a new technique of manual ambu-bag ventilation during nonbronchoscopic bronchoalveolar lavage (BAL) in critically ill premature infants dependent on mechanical ventilation. Seventeen infants underwent 34 BALs. Each patient underwent one, two, or three BALs as part of an ongoing study of the effect of dexamethasone therapy on inflammatory mediators in the lung in patients with respiratory distress syndrome (RDS) or bronchopulmonary dysplasia (BPD). All patients were < 30 weeks of gestational age; 11 of 17 patients were < 1 month old at the time of the first BAL. There were no major complications, such as lung abscess, pulmonary hemorrhage, or pneumothorax. Pooled results from BALs on days 0, 3, and 10 of dexamethasone therapy (n= 34), as well as results from each separate day, showed no significant difference in oxyhemoglobin saturation (Spo2), heart rate, or systolic blood pressure (SBP) before compared to after the procedure. After 10 of 34 BALs, SBP increased or decreased by ≥10 Torr and after 2 of 34 BALs, Spo2fell >5%, to a value of < 90%. After 2 of 34 BALs (6%), subjects did not return to baseline ventilator settings for >24 h. BAL sample data showed a mean of 41% macrophages in the cell count differentials. Mean BAL return was 45% of the fluid instilled. At follow-up 2 to 20 months after BAL, 16 of 17 patients were alive. One patient died of sepsis and severe RDS/BPD 6 days after BAL. We conclude that our technique of non-bronchoscopic catheter BAL using manual ambu-bag ventilation was generally well tolerated and sampled the alveolar space; however, critically ill, mechanically ventilated premature infants may require increased respiratory support after the procedure.