Photopolymerization of diacetylene-containing amphiphiles in substrate-supported bilayers has been studied in connection with the development of a new fabrication strategy of micropatterned biomimetic membrane systems. Two types of amphiphilic diacetylene molecules were compared, one being a monoalkyl phosphate, phosphoric acid monohexacosa-10,12-diynyl ester (1), and the other being a phospholipid, 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine (2). The bilayers of monomeric diacetylene amphiphiles were deposited onto substrates by the Langmuir-Blodgett/Langmuir-Schaefer methods. Both amphiphiles could be polymerized successfully on oxide and polymer-coated substrates. However, the phospholipid (2) showed a markedly higher mechanical stability of the polymerized bilayer films compared with the monoalkyl phosphate (1). Micropatterns were imposed in the bilayers by using a physical mask to protect the monomeric lipids partially upon UV irradiation. In the case of 2, monomeric bilayers could be removed selectively by ethanol after the lithographic photopolymerization, resulting in the formation of wells between the polymerized bilayers. These wells were filled with fluid phosphatidylcholine bilayers by fusion of vesicles. The rigid polymeric bilayer and the fluid biomimetic bilayer could be integrated as a composite bilayer membrane with defined spatial patterns, offering new possibilities to construct complex and versatile biomimetic membrane systems.