Phosphatidylethanolamine (PE) and phosphatidylgycerol (PG) are the main components of tile inner membrane of Escherichia coli. Mixtures of PE and PG mimicking the proportions found in E. coli have been extensively used to reconstitute transmembrane proteins as lactose permease (LacY) in proteoliposomes because in this environment the protein shows maximal activity. Hence, the study of the physicochemical properties of this phospholipid matrix becomes of potential interest. In previous Studies, 1,2 we used atomic force microscopy (AFM) and force spectroscopy (FS) to Study tile topographic and nanomechanical properties of supported lipid bilayers (SLBs) of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphothanolamine (POPE) and of POPE and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) (3:1, mol/mol). The study reported here was extended for completeness to asymmetric SLBs obtained by the Langumuir-Blodgett (LB) method. Thus, we prepared SLBs with the proximal leaflet extracted at 30 mN.m(-1) and the distal leaflet extracted at 25 mN.m(-1). We prepared SLBs with both leaflets with same composition (POPG/POPG), and also with the proximal leaflet of POPE and tile distal leaflet of POPG or POPE:POPG (3: 1, mol/mol). The topography of the SLBs acquired in liquid was compared with the topography of the monolayers acquired in air. Breakthrough (Fy) and adhesion forces (F-adh) of SLBs were extracted from force curves. The values obtained are discussed in terms of tile possible involvement of the nanomechanical properties of the SLBs in membrane protein insertion. The results provide means for the observation that insertion of LacY in POPE:POPG (3:1, mol/mol) occurs preferentially in the fluid phase, which is the phase with the lower FY and tile higher F-adh.