Bilayer lipid membranes (BLMs) can be formed on hydrophilic cushions that are tethered to a surface. Such tethered BLMs (tBLMs) are well suited to study the functional characteristics of membrane proteins (e.g., ion channels) because they (1) provide an ion reservoir between the surface and the BLM, which is necessary for electrochemical characterization, and (2) prevent contact between extra-membrane portions of the protein and the surface, thus allowing natural protein conformation and movement. We have created tBLMs on semi-transparent gold surfaces in microfluidic channels using phosphatidylthioethanol, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine- N-[3-(2-pyridyldithio)propionate], and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[PDP(polyethylene glycol)2000] as anchoring lipids and 1,2-dioleoyl-sn-glycero-3-phosphocholine as the free lipid. The tBLMs were characterized using fluorescence recovery after pattern photobleaching (FRAPP) to obtain diffusion coefficients and mobile fractions. The effect of molecular structure of the tethering lipids on the diffusion coefficient and mobility was investigated.