Addition of ionic and nonionic polymers can improve the function of therapeutic surfactants in vitro and in vivo, especially under conditions that tend to inhibit surfactant activity. Since surfactant proteins also act to reduce surfactant inhibition, we studied the relative effects of a synthetic peptide (that mimics some of the properties of a surfactant protein), polymers, and their combination on function of surfactant phospholipid activity in vitro. We evaluated surface activity after adding polymers-polyethylene glycol or hyaluronan-to a lipid mixture with or without the synthetic peptide, sinapultide (KL(4)). Using a pulsating bubble surfactometer, we measured peptide/polymer effects separately or combined at two peptide concentrations. Phospholipid mixtures, with or without KL(4) or polymers, all demonstrated good surface activity. With serum present as an inhibiting agent, adding either concentration of KL(4) reduced inhibition. Mixtures containing the higher concentration of KL(4) required higher concentrations of serum for inhibition to occur. Adding either polymer to mixtures with KL(4) further decreased susceptibility to inhibition (required higher serum concentrations). In the presence of serum, high molecular weight hyaluronan with KL(4) at 0.4 mg/ml improved surface activity to a greater degree than 0.8 mg/ml KL(4) without polymer. If the beneficial effects of adding polymer to KL(4)-lipid mixtures are also borne out in the treatment of experimental lung injury, these peptide-polymer surfactant combinations may eventually prove useful in the treatment of some forms of acute lung injury in humans.