Clinical deterioration has been observed in sick neonates during lumbar puncture. This study was done to determine if hypoxemia occurred during lumbar puncture, if hypoxemia was position dependent, if transcutaneous PO2 (TcPO2) monitoring effected hypoxemia, and what possible mechanisms were involved. Twenty-six neonates received lumbar punctures in either a standard lateral knee-chest position, sitting position, or modified lateral without knee-chest position. Care was taken not to extend or flex the neck. Mean TcPO2 was lower for standard lateral than sitting or modified lateral positions. The time TcPO2 was under 50 mm Hg was greater for standard lateral positions than sitting or modified lateral positions. Increased intraesophageal pressure, in the standard lateral position, suggests extrathoracic compression of the chest by the abdominal contents. We recommend lumbar punctures be done in the sitting or modified lateral position.