Ventilation style influences lung injury and the amount of large-aggregate biophysically active surfactant in adult lungs. We asked how positive end-expiratory pressures (PEEP) would influence clinical responses and surfactant pools in surfactant-treated preterm lambs ventilated for 7 h with tidal volumes (VT) of 10 ml/kg. The 126-d gestation preterms were delivered and treated with 100 mg/kg recombinant human surfactant protein C (rSP-C) containing surfactant and ventilated with zero, 4, or 7 cm H(2)O of PEEP. A comparison group was treated with natural sheep surfactant and ventilated with zero PEEP. Physiologic measurements were similar for lambs treated with rSP-C surfactant and natural surfactant. PEEP 4 and 7 improved oxygenation and compliance relative to either group of lambs ventilated with PEEP zero. The maximal lung volumes measured at 40 cm H(2)O pressure after 7 h ventilation for the PEEP 4 and 7 groups were more than double those measured for either PEEP zero group. Alveolar surfactant pools were larger for the PEEP 7 group, and the large-aggregate fraction was increased for the PEEP 4 and 7 groups, resulting in large-aggregate pool sizes that were 3-fold higher for the PEEP 4 and 4-fold higher for the PEEP 7 groups relative to the PEEP zero group treated with rSP-C surfactant. All large-aggregate surfactants lowered minimal surface tensions of a captive bubble to less than 5 mN/m. In preterm surfactant-treated lambs PEEP improved lung function and maintained more of an rSP-C surfactant in the biophysically active form.