The localization of S-antigen in ultra-cryosections of rat retina has been studied and compared with that of opsin by immunoelectron microscopy. Monospecific antisera to both proteins, and Protein A-coated gold particles of two sizes were used for (double) labeling. In the light-adapted retina, S-antigen showed codistribution with opsin in the rod outer segments. S-antigen labeling was diminished in dark-adapted retina. In retina pigment epithelium, multi-layered bodies were found containing S-antigen and rhodopsin representing spent rod outer segment fragments. In addition to theoretical considerations, there is indication that S-antigen is closely associated with the cytoplasmic surface of rod outer segment disc membranes. It seems moreover likely that S-antigen and opsin are closely associated in the illuminated retina. An important reason for this assumption was the observation that double labeling experiments showed interference of the antibodies to both antigens probably due to steric hinderance. The present results are complementary to, and in agreement with biochemical data from literature and point to an important function of S-antigen in the process of phototransduction.