ABSTRACT The first step of vision takes place in the retina where light is captured by the outer segment organelle of rod and cone photoreceptors. The outer segment is a modified primary cilium that contains a stack of flat disc-shaped membranes. This structural arrangement maximizes light detection by forcing photons to pass through hundreds of membrane layers densely packed with the light absorbing protein, opsin. A byproduct of light absorption is photooxidative damage, which the photoreceptor overcomes by continuously replacing its entire membrane-rich outer segment with new protein and lipid material. Defects in outer segment trafficking, formation, and function ultimately result in photoreceptor cell loss and cause a broad spectrum of inherited retinal dystrophies including retinitis pigmentosa, cone-rod dystrophy and Stargardt disease. It is now well established that photoreceptors utilize multiple trafficking pathways to deliver structural and signaling proteins to the light-sensitive outer segment organelle; however, how these pathways are coordinated, the molecular processes regulating delivery, why a cargo chooses a specific route remains largely unknown. The goals of our proposal are to investigate the trafficking pathways of outer segment membrane cargos that reside within the discs (rhodopsin), the disc rims (peripherin-2 and ABCA4) and the plasma membrane surrounding the discs (CNG channel). This comprehensive approach will allow us to understand shared and divergent features between specific trafficking pathways as well as build a complete overview of intracellular routes to the outer segment. Our aims utilize proximity labeling approaches to uncover transport partners in vivo as well as gene delivery techniques to rapidly screen candidates for targeting defects and uncover signals governing conventional and unconventional delivery to the outer segment in mouse rods. Completion of our aims will provide insight into how outer segment trafficking pathways cooperate to deliver the structural and functional proteins necessary to form the light-sensing outer segment organelle as well as how these processes fail during disease. Looking forward, our proposal aims to provide fundamental knowledge about membrane trafficking to sensory cilia in general as well as help to inform future therapeutic strategies for patients suffering from inherited blindness.