Fifty formalin-fixed and paraffin-embedded retinoblastoma specimens were studied for the presence of S-100 protein in malignant tumour cells and in reactive glial cells. Its distribution in four normal human eyeglobes enucleated because of an orbital tumour was also studied. The laboratory method was a sensitive immunohistochemical staining procedure, and sections from human brain served as controls. Three anti-S-100 protein antisera were used to determine whether previously reported discrepancies could be related to differences in antibody specificity, but all gave identical results. In morphologically normal human retina, a strong reaction for S-100 protein was seen in astrocytes, and a faint, inconsistent positivity was detected in Müller's cells. All intraocular retinoblastomata studied contained cells positive for S-100 protein, which seemed to be reactive glial cells derived from infiltrated retina. Undifferentiated malignant retinoblastoma cells, as well as differentiated tumour cells in Flexner-Wintersteiner rosettes were negative for S-100 protein. Differentiated tumours appeared to contain more S-100 protein-positive glial cells than undifferentiated tumours, but this may be related to their different growth rates. The results do not support the theory that retinoblastoma cells express glial properties.