Monoclonal antibodies were used with radioimmune assays and immunocytochemical techniques to identify and localize membrane proteins in bovine rod outer segment (ROS) disc membranes. When ROS membrane proteins were separated by SDS-polyacrylamide gel electrophoresis in the presence of the sulfhydryl reducing agent, 2-mercaptoethanol, two monoclonal antibodies designated as 3B6 and 2B6 were found to bind to a polypeptide having an apparent molecular weight (Mr) of 33,000 daltons. In the absence of 2-mercaptoethanol, these monoclonal antibodies bound to a doublet having Mr of 67,000 and 69,000. Immunogold-dextran labeling of ROS sections embedded in Lowicryl resin indicated that this protein is localized around the periphery of the ROS organelle where the discs come in close contact to the ROS plasma membrane. Immunogold labeling of morphologically intact isolated discs prepared by mild trypsinization of ROS fragments confirmed that this disc membrane protein is localized along the rim region of discs. On the basis of these localization studies, the authors have named this protein peripherin. Immunogold-dextran markers were also used with previously characterized antirhodopsin monoclonal antibodies to visualize the distribution of rhodopsin on isolated discs. Dense labeling was observed along the lamellar region of the discs, but little if any labeling was observed on the extreme edges of the discs. These results are consistent with the view that the lamellar region of discs containing rhodopsin is a distinct membrane domain from the rim region which contains peripherin, a high Mr rim protein and possibly other proteins involved in disc-disc and disc-plasma membrane interactions.