The subcellular localization of three photoreceptor antigens (RET-P1, rhodopsin and RET-P2) has been studied by electron microscopic immunocytochemistry of rat retinas. Localization was also examined by determining the amount of RET-P1 and RET-P2 antigen in various subcellular fractions. RET-P1 and RET-P2 antigens were further characterized by immunoblotting of crude retina membrane proteins which had been separated by one-dimensional gel electrophoresis. RET-P1 antigen has been detected with a monoclonal antibody that reacts with the perikarya, inner segments, and outer segments of adult rat photoreceptors by peroxidase immunolabelling of fixed tissue sections. Analysis at the electron microscopic level has shown that RET-P1 antigen is located on the external face of the inner and outer segment plasma membrane. A monoclonal antibody against purified bovine rhodopsin (RHO-C7) labels the outer segments of rat retinas by peroxidase immunocytochemistry. Ultrastructural antibody localization indicates that this particular determinant of rhodopsin is exposed on the external face of the plasma membrane of outer segments and may also be expressed on the surface of the inner segments. RET-P2 antibody labels only the outer segments of adult rat photoreceptors by peroxidase immunocytochemistry. The light microscopic labelling of RET-P2 antibody in the presence, but not in the absence, of detergent suggests that it is an intracellular antigen. The results of both ultrastructural labelling and biochemical fractionation are consistent with the localization of RET-P2 antigen on the internal face of the plasma membrane and/or the cytoplasmic face of the disc membranes. RET-P2 antigen was found to be a protein (or glycoprotein) of apparent molecular weight 38 000 +/- 3000.