Spleen cells of BALB/c mice, previously immunized with bovine retinal S-antigen, were fused with Sp2/0-Ag14 mouse myeloma cells. Two monoclonal antibodies (MAbs) MAbA9-C6 (IgG2a isotype) and MAbA1-G5 (IgG1 isotype) were selected on the basis of reactivity in an ELISA and immunofluorescent assay. In radioimmunoassay MAbA9-C6 and MAbA1-G5 do not compete and appear to define unrelated epitopes of S-antigen. Both MAbs reacted in the ELISA assay, whereas only MAbA9-C6 bound to S-antigen in fixed tissue sections. Because MAbA9-C6 was useful for immunocytochemistry, it was studied in more detail. MAbA9-C6 bound to all vertebrate retinas tested including human, bovine, guinea pig, and mice. The immunoreactivity of MAbA9-C6 also was studied in the developing rat retina and pineal gland. In the morphologically undifferentiated retina, assessed by conventional light microscopy, there was an incomplete separation of the outer neuroblastic cells. However, in the same retina a distinct zone, corresponding to S-antigen immunoreactivity, was present indicating antigenic differentiation with regard to S-antigen at this stage of retinal development. In the pineal gland, S-antigen immunoreactivity was first observed in the three day old rat and at all stages examined thereafter. The usefulness of these two MAbs in the study of the embryologic development of the retina and of the antigenic epitopes of S-antigen is discussed.