Phospholipid bilayers were studied by means of atomic force microscopy (AFM) and a surface force apparatus (SFA). The stability of the supported bilayers was described by the amount of irregularities in the topography of the membrane by means of AFM and by the occurrence of hemifusion in the SFA, which is an indicator of defective bilayers. The bilayers, composed of lipids having the same headgroup but different chain lengths in the two leaflets, were prepared by Langmuir-Blodgett deposition and transferred at different surface pressures. The topography of the supported bilayers in aqueous solution, as imaged by AFM, revealed an increasing number of defects in the supported lipid membranes with decreased deposition pressure of the outer lipid layer. These defects, which appeared in the form of monolayer and bilayer (self-assembled) thick holes within the membrane, were energetically favorable over an evenly depleted bilayer. We found that the quantity of these defects (holes of </=0.5 micro m diameter and covering up to 30% of the surface area) correlated well with the stability of the bilayers as measured by SFA, a truly complementary instrument.