Available surfactants for treatment of respiratory distress syndrome in newborn infants are derived from animal lungs, which limits supply and poses a danger of propagating infectious material. Poly-Valpoly-Leu analogues of surfactant protein (SP)-C can be synthesized in large quantities, and exhibit surface activity similar to SP-C. Here, activity of synthetic surfactants containing a poly-Leu SP-C analogue (SP-C33) was evaluated in ventilated premature newborn rabbits. Treatment with 2.5 ml/kg body weight of 2% (w/w) SP-C33 in 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), 68:0:31, 68:11:20 or 68:16:15, (w/w), suspended at 80 mg/ml gave tidal volumes (V_T) of 20-25 ml/kg body weight with an insufflation pressure of 25 cmH₂O and no positive end-expiratory pressure (PEEP), comparable to the V_T for animals treated with the porcine surfactant Curosurf®. Non-treated littermates had a V_T of about 2 ml/kg body weight. The V_T for SP-C33 in DPPC/egg phosphatidylglycerol (PG)/palmitic acid (PA), 68:22:9 (w/w), DPPC/POPG/PA, 68:22:9 (w/w), and DPPC/POPC/POPG, 6:2:2 (w/w) was 15-20 ml/kg body weight. Histological examination of lungs from animals treated with SP-C33-based surfactants showed incomplete, usually patchy air expansion of alveolar spaces associated with only mild airway epithelial damage. Lung gas volume after 30 min of mechanical ventilation were more than 3-fold larger in animals treated with Curosurf than in those receiving SP-C33 in DPPC/POPC/POPG, 68:11:20. This difference could be largely counterbalanced by ventilation with PEEP (3-4 cm H₂O). An artificial surfactant based on SP-C33 improves tidal volumes in immature newborn animals ventilated with standardized peak pressure, but requires PEEP to build up adequate lung gas volumes.