<i>Background:</i> Modified natural surfactants currently used for treatment of respiratory distress syndrome contain about 0.5–1% (w/w phospholipids) of each of the surfactant proteins SP-B and SP-C. The supply of these preparations is limited and synthetic surfactant preparations containing lipids and peptides are under development. <i>Objectives:</i> To investigate the potential of different concentrations of the SP-C analogue SP-C33 in 1,2-dipalmitoyl-<i>sn</i>-glycero-3-phosphocholine/1-palmitoyl-2-oleoyl-<i>sn</i>-glycero-3-phosphoglycerol (68:31, w/w). <i>Methods:</i> Surface activity was evaluated in pulsating and captive bubble surfactometers and in immature newborn rabbits. <i>Results:</i> Preparations containing ≧1% SP-C33 achieve minimum surface tension <5 mN/m indicating good biophysical activity, and increase tidal volumes in premature rabbit fetuses to the same level as a modified natural surfactant preparation does. Alveolar patency at end expiration, as evaluated by measurement of lung gas volumes, histological assessment of alveolar expansion and determination of alveolar volume density, was lower in the animals treated with synthetic surfactant than in those receiving modified natural surfactant. <i>Conclusions:</i> These data suggest that SP-C33 is similarly efficient as the native peptide in improving surface properties of phospholipids mixtures and in increasing lung compliance in surfactant-deficient states, but that other components are needed to maintain alveolar stability at low airway pressures.