The effect of exogenous cholesterol on the stability of surface films at 37 degrees C from various surfactants was studied with the pulsating bubble surfactometer. Addition of cholesterol (5%, w/w) to bovine lipid extract surfactant (bLES) or mixtures of dipalmitoylphosphatidylcholine/1-palmitoyl-2-oleoyl-phosphatidylglycerol /SP-B (7:3:1%) dispersed in 1.5 mM CaCl2/0.9% NaCl resulted in unstable surface films. Although 10% cholesterol only partially impaired the surface activity of bLES, it virtually abolished that of the reconstituted surfactant. The inhibitory effects of cholesterol were significantly repressed by SP-A (10%, w/w of lipid) and 3 mM CaCl2 or 5 mM CaCl2 without SP-A. Adsorption of cholesterol from various surfactants into the air/water interface was examined by measuring the surface radioactivity of [14C]cholesterol. Cholesterol alone dispersed in 1.5 mM CaCl2/0.9% NaCl could not adsorb to the interface, but it adsorbed readily when mixed with bLES. Cholesterol adsorption was markedly suppressed by SP-A in 3 mM CaCl2/0.9% NaCl or 5 mM CaCl2/0.9% NaCl without SP-A. Electron microscopy revealed striking ultrastructural differences between bLES/5% cholesterol/10% SP-A in 3 mM CaCl2/0.9% NaCl and bLES/5% cholesterol in 3 or 5 mM CaCl2/0.9% NaCl. The former exhibited large multilayer and small unilamellar vesicles, while the latter displayed condensed patches of aggregates. Adsorption studies showed aggregated patches adsorbed more rapidly than vesicles but attained lower equilibrium surface pressures. These results indicate SP-A and calcium limit the adsorption of surfactant cholesterol to the air-water interface.