Free-standing lipid membranes are promising as artificial functional membrane systems for application in separation, filtration, and nanopore sensing. To improve the mechanical properties of lipid membranes, UV-polymerized lipids have been introduced. We investigated free-standing as well as substrate-supported monolayers of 1-palmitoy1-2(10,12-tricosadiynoyl)-sn-glycero-3-phosphoethanolamine (PTPE) and 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine (DiynePC) and characterized them with respect to their structure, morphology, and stability. Using helium ion microscopy (HIM), we were able to visualize the integrity of the lipid 2D-nanomembranes spanning micrometer-sized voids under high-vacuum conditions. Atomic force microscopy (AFM) investigations under ambient conditions revealed formation of intact and robust pore-spanning 2D-nanomembranes up to 8 X 2 mu m(2) in size. Analysis by attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) verified a distinct reduction of signal at 2143 cm(-1) from diacetylene groups in the 2D-nanomembranes after UV-polymerization. Further high resolution AFM investigations of unpolymerized lipid monolayers revealed a well-ordered two-dimensional network, when deposited on highly oriented pyrolytic graphite (HOPG). These structures were inhibited for polymerized adlayers. Structural models for the molecular arrangement of the adlayers are proposed and discussed.