We report on a simple method to generate bilayer membranes on a smooth layer of crystalline bacterial cell surface (S-layer) protein lattices, deposited on a microfiltration membrane (MFM), the so-called S-layer ultrafiltration membrane (SUM). Folded bilayers, MFM-, and SUM-supported lipid membranes showed a specific capacitance of 0.6−0.7 μF/cm2. The S-layer produced a stabilizing effect on the lipid membrane; folded and MFM-supported membranes ruptured at the application of a first and a second voltage ramp, respectively, whereas the SUM-supported membranes could withstand multiple voltage ramps. The lifetime of the lipid membranes increased significantly in the order MFM-supported membrane < folded membrane < SUM-supported membrane. By contrast with MFM-supported lipid membranes, successful reconstitution of staphylococcal α-hemolysin (αHL) was observed in both folded and SUM-supported membranes. The unitary conductance of an αHL pore was similar when reconstituted in folded and in SUM-supported lipid ...