Background: Retinoblastoma is the most common childhood tumor of the eye, arising from cells that are defective in both copies of the retinoblastoma susceptibility gene (RB1). Most retinoblastoma tumor cells eventually undergo programmed cell death (i.e., apoptosis); however, some cells can acquire the ability to metastasize and become immortal. Transfection of immortal retinoblastoma cells with DNA sequences encoding wild-type p53 protein induces cell death, suggesting that the loss of both RB1 and p53 functions may be required for cell immortalization. We have examined this possibility by characterizing the p53 protein and messenger RNA in six independently isolated, immortalized retinoblastoma cell lines. Methods: Western blotting methods were used to assess p53 protein level in each cell line, and Cleavase Fragment-Length Polymorphism analysis of complementary DNAs was used to screen for mutations in p53 messenger RNA. Localization of p53 protein in cells of the immortalized lines and in specimens of retinoblastoma tumors was achieved by means of indirect immunofluorescence and immunocytochemistry, respectively. Results: All six immortalized cell lines expressed wildtype p53 messenger RNA and high levels of p53 protein. Although p53 is normally a nuclear protein, the p53 in four of the six cell lines was located predominately in the cytoplasm; in the remaining two cell lines, p53 was localized in both the nucleus and the cytoplasm. Cytoplasmic localization of p53 in retinoblastoma tumor specimens was rare and usually restricted to cells that had invaded adjacent ocular tissues, indicative of the early stages of metastasis. Conclusions: Some immortalized retinoblastoma cells may exhibit p53 dysfunction through nuclear exclusion of wild-type p53 protein.