Mixed self-assembled monolayers (SAMs) of beta-mercaptoethanol and the new synthetic lipid 1,2-dipalmityl-3-[w-mercaptonona(ethylene oxide)] glycerol (FC 16) were investigated for their ability to form sparsely tethered bilayer lipid membranes (stBLMs) completed with various phospholipids. We investigated the structural and functional properties of FC16-based stBLMs and compared these to stBLMs prepared using a previously characterized synthetic lipid, 1,2-dimyristyl-3-[omega-mercaptohexa(ethylene oxide)] glycerol (WC14). FC16-based stBLMs show increased resistivity to ion transfer and an increase in the submembrane space of approximately 0.5 nm. Importantly, FC16-based stBLMs formed well-defined, complete bilayers with charged phospholipids such as 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG). In these, POPG incorporates into the outer monolayer leaflet in the same ratio as in the immersion solution but is excluded from the inner leaflet. In all cases that we have investigated thus far, the area densities of the lipids within the bilayers were on average close to those in free bilayer membranes. For charged phospholipids, FC16 appears to provide a distinct advantage over WC14 for the formation of well-defined stBLMs.