The present paper describes a new and reproducible method to generate stable lipid membranes on porous supports. The support was produced by the deposition of a smooth layer of crystalline bacterial cell surface (S-layer) protein lattices on a microfiltration membrane (SUM). The commercially available main phospholipid of Thermoplasma acidophilum (MPL), a membrane-spanning tetraether lipid, but also mixtures of MPL with diphytanoylphosphatidylcholine (DPhPC) at molar ratios of MPL/DPhPC = 1:1 and 5:1 and pure DPhPC were spread at the air/water interface. The monomolecular films were transferred by one (MPL and mixtures) or two (DPhPC steps on the SUM. In addition, folded membranes were generated with DPhPC and the lipid mixtures. The specific capacitance of the tight SUM-supported membranes increased continuously with increasing MPL to DPhPC ratio from 0.62 muF/cm(2) (pure DPhPC) over 0.66muF/cm(2) (equimolar DPhPC/MPL)to 0.76 muF/cm(2) for pure MPL membranes. SUM-supported MPL membranes showed a lifetime of 8.3 +/- 2.9 h. An additional monomolecular S-layer protein lattice recrystallized on the lipid-faced side increased the lifetime significantly to 21.2 +/- 3.1 h. To prove the functionality of the membranes, gramicidin was reconstituted. All folded and SUM-supported membranes allowed reconstitution of gramicidin, and high-resolution measurements on single pores could be performed.