In this article, we describe the effect of a highly hemolytic saponin, digitonin, on model lipids cholesterol and dipalmitoylphosphatidylcholine (DPPC) using a combination of tensiometric (surface pressure and dilatational surface elasticity), spectroscopic (infrared reflection absorption spectroscopy, IRRAS), microscopic (fluorescence microscopy), and scattering techniques (neutron reflectivity, NR, and grazing incidence X-ray diffraction, GIXD). The monolayers of individual lipids and their 10:9 (mol/mol) mixture were exposed to an aqueous solution of digitonin (10(-4)M) by subphase exchange using a setup developed recently in our laboratory. The results confirm that digitonin can adsorb onto both bare and lipid-covered water air interfaces. In the case of DPPC, a relatively weak interaction can be observed, but the presence of cholesterol drastically enhances the effect of digitonin. The latter is shown to dissociate the weak cholesterol DPPC complexes and to bind cholesterol in an additional layer attached to the original lipid monolayer.