The effects of the reactive oxygen species (ROS) superoxide anion (O2*-) and hydroxyl radical (*OH) on the surface tension lowering properties of bovine lipid extract surfactant (BLES) were compared to the effects of calf serum protein (CSP) in a captive bubble surfactometer (CBS). O2*- was generated from xanthine/xanthine oxidase (X/XO), and *OH was generated by the Fenton reaction. ROS were demonstrated by electron spin resonance (ESR) using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as the spin trap. Lipid peroxidation was measured using the thiobarbituric acid method. *OH had broad inhibitory effects on surface tension parameters, including adsorption, minimum surface tension, percentage film area change and film compressibility. O2*- showed inhibitory effects on adsorption, film area change and film compressibility but had no significant effect on minimum surface tension. Both O2*- and *OH treatment were associated with a large 'squeezeout' plateau around 20-25 mN/m in the surface tension-area relation, indicating poor film organization during the compression phase. At the concentrations used, ROS were associated with lipid peroxidation of BLES, which also demonstrated radical scavenging properties. Calf serum protein produced inhibitory effects on adsorption, minimum surface tension and percentage film area change that were quantitatively similar to those produced by *OH. The effects on film compression were significantly greater and qualitatively different from those seen with either O2*- or *OH. We conclude that the inhibition of BLES surface activity by ROS and inhibitory proteins can be distinguished in the captive bubble surfactometer and, particularly, by changes in the film compressibility modulus.