The inhibitory effects of reactive oxygen species (ROS) on the surface tension-lowering abilities of three surfactants were compared: natural lung surfactant (NLS), KL4 surfactant containing synthetic peptide resembling the hydrophobic/hydrophilic domains of SP-B in an aqueous dispersion of phospholipids, and Survanta (SUR) containing SP-B and SP-C. The inhibitory concentrations of Fenton reactants (i.e. 0.65 mM FeCl2, 0.65 mM EDTA, 30 mM H2O2), deduced from dose-response plots of FeCl2 on minimum surface tension (MST) of SUR, were used to assess the Fenton effect on biophysical properties of various surfactants. Neither H2O2 (30 mM) nor FeCl2 with EDTA (both 0.65 mM) alone affected surfactant function, but when mixed together significantly increased (p < 0.01) the MST of SUR compared with KL4 (p < 0.05) in a FeCl2 concentration-dependent manner. This effect on NLS was not significant (p = 0.05) at similar phospholipid concentrations. Also, the range of increases in surface adsorption in mN/m at equilibrium surface tension (EST) was 27-40 for SUR, 36-44 for KL4, and 24-25 for NLS. We speculate that the presence of SP-A and the catalase content in NLS may have protective effects on inactivation of NLS by ROS. We conclude that the in vitro Fenton effect could be a valuable test system for comparing the inactivation range of surfactants by oxyradicals.