The factors regulating lung aeration and the initiation of pulmonary gas exchange at birth are largely unknown, particularly in infants born very preterm. As hydrostatic pressure gradients may play a role, we have examined the effect of a positive end-expiratory pressure (PEEP) on the spatial and temporal pattern of lung aeration in preterm rabbit pups mechanically ventilated from birth using simultaneous phase contrast X-ray imaging and plethysmography. Preterm rabbit pups were delivered by caesarean section at 28d of gestational age, anesthetized, intubated and placed within a water-filled plethysmograph (head out). Pups were imaged as they were mechanically ventilated from birth with either a PEEP of 0 cmH₂O or 5 cmH₂O. The peak inflation pressure was held constant at 35 cmH₂O. Without PEEP, gas only entered into the distal airways during inflation. The distal airways collapsed during expiration and as a result, the functional residual capacity (FRC) did not increase above the lung's anatomical dead space volume (2.5±0.8 mL/kg). In contrast, ventilation with 5 cmH₂O PEEP gradually increased aeration of the distal airways, which did not collapse at end expiration. The FRC achieved in pups ventilated with PEEP (19.9±3.2 mL/kg) was significantly greater than in pups ventilated without PEEP (-2.3±3.5 mL/kg). PEEP greatly facilitates aeration of the distal airways, the accumulation of FRC and prevents distal airway collapse at end-expiration in very preterm rabbit pups mechanically ventilated from birth.