Membrane fusion is defined as the consolidation of two membrane bilayers and a subsequent mixing of the two previously separated aqueous compartments. Membrane fusion processes are mediated and regulated by a growing family of soluble and integral membrane proteins termed fusion proteins.1,2 Fusion proteins share structural and functional properties and aid in the thermodynamically unfavorable fusion event by promoting hydrophobic interactions that favor fusion.1–3 Within retinal photoreceptor rod cells membrane fusion is a component step of at least three essential cellular processes. Fusion is necessary for the delivery of proteins and lipids in vesicles from the rod inner segment (site of synthesis) to the rod outer segment (ROS). Two additional fusion processes preserve the unique architecture of the outer segment by maintaining the outer segment at a constant length. The coordinated processes of disk morphogenesis and compensatory disk shedding4 require the fusion of two opposing membranes: the fusion of two outgrowing inside-out disk rims for morphogenesis,5 and disk–plasma membrane fusion for disk packet formation.6 Fusion during disk packet formation is documented in microscopy studies in which an analysis of dye penetration into distinct regions of the ROS found that large molecules do not enter the narrow bands of the dye-stained region of the ROS, suggesting a fusion of the plasma membrane with the disk membranes.7,8 This fusion is mediated by a fusion protein unique to photoreceptors: peripherin/rds. In this chapter we describe the protocols used in photoreceptor cell-free fusion assays and the characterization of peripherin/rds as a rod cell-specific fusion protein