The rate of change of surface pressure, pi, in a Langmuir trough following the deposition of surfactant suspensions on subphases containing serum, with or without polymers, is used to model a likely cause of surfactant inactivation in vivo: inhibition of surfactant adsorption due to competitive adsorption of surface active serum proteins. Aqueous suspensions of native porcine surfactant, organic extracts of native surfactant, and the clinical surfactants Curosurf, Infasurf, and Survanta spread on buffered subphases increase the surface pressure, pi, to approximately 40 mN/m within 2 min. The variation with concentration, temperature, and mode of spreading confirmed Brewster angle microscopy observations that subphase to surface adsorption of surfactant is the dominant form of surfactant transport to the interface. However (with the exception of native porcine surfactant), similar rapid increases in pi did not occur when surfactants were applied to subphases containing serum. Components of serum are surface active and adsorb reversibly to the interface increasing pi up to a concentration-dependent saturation value, pi(max). When surfactants were applied to subphases containing serum, the increase in pi was significantly slowed or eliminated. Therefore, serum at the interface presents a barrier to surfactant adsorption. Addition of either hyaluronan (normally found in alveolar fluid) or polyethylene glycol to subphases containing serum reversed inhibition by restoring the rate of surfactant adsorption to that of the clean interface, thereby allowing surfactant to overcome the serum-induced barrier to adsorption.