Preparation by the developing alveolar epithelium for the transition to air breathing and surfactant secretion at birth are critical components of neonatal survival. We combined morphometric analysis and biochemical assays of lung phospholipids to measure the amount and redistribution of lung surfactant during the perinatal period of rats. Within 10 min of the start of air breathing, there was a small increase in type II cell lamellar body content by morphometric and biochemical estimates. By 24 h, the whole lung and alveolar extracellular pool surfactant lipid had substantially increased. Subfractionation of the alveolar surfactant pool obtained at four times, from birth to 24 h of life, demonstrated a 20-fold increase in the ratio of phospholipid in a tubular myelin-rich fraction compared to a unilamellar vesicle-rich fraction. We conclude that packaging of surfactant may be very active immediately postbirth. Our results also indicate a major shift in the physical forms of extracellular surfactant during the first hours of air breathing.