Fifty-four formalin-fixed and paraffin-embedded intraocular retinoblastoma specimens and three human eyes enucleated because of orbital tumors were studied for the presence of synaptophysin, a neuron-associated integral membrane glycoprotein of presynaptic vesicles, by using the monoclonal antibody SY38. Normal human brain was used as control. In the human retina, synaptophysin-like immunoreactivity was present in both plexiform layers, but could not be detected in neuronal perikarya. However, in reactive retinas present in retinoblastoma eyes, synaptophysin was often observed in perikarya and processes of photoreceptors. Positive neoplastic cells were found in 45 of the 54 retinoblastomas. Differentiated tumors tended to contain greater numbers of positive cells than undifferentiated ones, a third of which were entirely negative. Identical immunoreactivity was seen in frozen specimens from human retina and from three retinoblastomas. Using Western blotting, a major polypeptide comigrating with human brain synaptophysin was detected in human retina, and a similar but slightly slower migrating band in retinoblastoma. The results support a primarily neuronal origin for this tumor and point to the possibility that synaptic elements, previously observed in a few cases, may be more frequent in retinoblastoma than had been thought.