The plasma membrane of animal cells is attached to the cytoskeleton, which significantly contributes to the lateral tension of the membrane. Lateral membrane tension has been shown to be an important physical regulator of cellular processes such as cell motility and morphology as well as exo- and endocytosis. Here, we report on lipid bilayers spanning highly ordered pore arrays, where we can control the lateral membrane tension by chemically varying the surface functionalization of the porous substrate. Surface functionalization was achieved by a gold coating on top of the pore rims of the hexagonal array of pores in silicon nitride substrates with pore radii of 600 nm followed by subsequent incubation with various n-propanolic mixtures of 6-mercapto-1-hexanol (6MH) and O-cholesteryl N-(8'-mercapto-3',6'-dioxaoctyl)carbamate (CPEO3). Pore-spanning membranes composed of 1,2-diphytanoyl-sn-glycero-3-phosphocholine were prepared by spreading giant unilarnellar vesicles on these functionalized porous silicon nitride substrates. Different mixtures of 6MH and CPEO3 provided self-assembled monolayers (SAMs) with different compositions as analyzed by contact angle and PM-IRRAS measurements. Site specific force-indentation experiments on the pore-spanning membranes attached to the different SAMs revealed a clear dependence of the amount of CPEO3 in the monolayer on the lateral membrane tension. While bilayers on pure 6MH monolayers show an average lateral membrane tension of 1.4 mN m(-1), a mixed monolayer of CPEO3 and 6MH obtained from a solution with 9.1 mol % CPEO3 exhibits a lateral tension of 5.0 mN m(-1). From contact angle and PM-IRRAS results, the mole fraction of CPEO3 in solution can be roughly translated into a CPEO3 surface concentration of 40 mol %. Our results clearly demonstrate that the free energy difference between the supported and freestanding part of the membrane depends on the chemical composition of the SAM, which controls the lateral membrane tension.