The outer segment is a specialized compartment of vertebrate rod and cone photoreceptor cells where phototransduction takes place. In rod cells it consists of an organized stack of disks enclosed by a separate plasma membrane. Although most proteins involved in phototransduction have been identified and characterized, little is known about the proteins that are responsible for outer segment structure and renewal. In this study we have used a tandem mass spectrometry-based proteomic approach to identify proteins in rod outer segment preparations as an initial step in defining their roles in photoreceptor structure, function, renewal and degeneration. Five hundred and sixteen proteins were identified including 41 proteins that function in rod and cone phototransduction and the visual cycle and most proteins previously shown to be involved in outer segment structure and metabolic pathways. In addition, numerous proteins were detected that have not been previously reported to be present in outer segments including a subset of Rab and SNARE proteins implicated in vesicle trafficking and membrane fusion. Western blotting and immunofluorescence microscopy confirmed the presence of Rab 11b, Rab 18, Rab 1b and RabGDI in outer segments. The SNARE proteins, VAMP2/3, syntaxin 3, NSF, and Munc 18 detected in outer segment preparations by mass spectrometry and western blotting were also observed in outer segments by immunofluorescence microscopy. Syntaxin 3 and NSF had a restricted localization at the base of the outer segments, while VAMP2/3 and Munc 18 were distributed throughout the outer segments. These results suggest Rab and SNARE proteins play a role in vesicle trafficking and membrane fusion as part of the outer segment renewal process. The dataset generated in this study is a valuable resource for further analysis of photoreceptor outer segment structure and function.