Specimens of 24 enucleated eyes from patients with retinoblastomas in situ were examined by labelled streptavidin biotin (LSAB) method to ascertain the distribution of S-100 protein and compared with 28 normal developing eyes. Polyclonal anti-S-100 antiserum and monoclonal α and β chain anti-S-100 antibodies were used in this study. In the normal retina a weak reaction was observed in astrocytes and ganglion cells at 24 gestational weeks (GW) and there was an increase in the intensity of staining with increasing age. In the morphologically normal retina with retinoblastoma, astrocytes, ganglion cells and Müller cells showed positive reactions for S-100 protein. Examined tumors showed S-100 protein-positive glial cells that were interpreted as reactive astrocytes. No reaction product was detected in the tumor cells. These findings support the concept that in the retinoblastoma, only glial cells are positive for S-100 protein.