Photopolymerization of the diacetylene-containing phospholipid 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine (1) in substrate-supported planar lipid bilayers (SPBs) has been studied by using multimode atomic force microscopy (AFM). Monolayers and bilayers of 1 have been transferred onto glass substrates by the Langmuir-Blodgett (LB) and Langmuir-Schaefer (LS) techniques, respectively. Bilayers of monomeric 1 were removed easily from the substrate surface by detergent solutions (0.08 M sodium dodecyl sulfate (SDS)). Once they were photopolymerized by UV light, the bilayers became resistant toward detergent solubilization or exposure to air. High-resolution AFM images revealed the molecular packing of the hydrocarbon tails and the headgroups for monolayers and bilayers, respectively. The lattice spacing of photopolymerized 1 bilayers was similar to that of monomeric bilayers, suggesting that the polymerization proceeds without disruption of the bilayer structure. Patterned SPBs of poly-1 were obtained by UV photolithography and selective removal of the protected monomeric bilayers. AFM revealed that the photopolymerization proceeds heterogeneously within the bilayers, depending on the UV irradiation dose. At longer UV exposure times, side reactions and/or reorganization of the molecules within the bilayer may occur.