From 49 eyes enucleated for retinoblastoma, two new cell lines, WERI-Rb24 (W-24) and WERI-Rb27 (W-27), were established in long-term culture (greater than 5 years). The W-24 cell line was derived from a 22-month-old boy with sporadic retinoblastoma; the W-27 cell line was derived from a 24-month-old boy with bilateral retinoblastoma. Both cell lines show abnormal mRNA transcripts corresponding to the retinoblastoma gene. At the DNA level, one retinoblastoma allele was not present in the W-24 cell line. In the W-27 cell line a mutation was identified in one allele, while the other appears grossly normal. Since no normal retinoblastoma mRNA can be detected in either cell line, a subtle mutation must occur on the grossly normal allele. Comparison of DNA in W-27 lymphocytes and tumor cells with DNA probe p6NR-0.5 indicates that the observed mutation occurred somatically. The application of these two new retinoblastoma cell lines to the characterization of defects in the retinoblastoma gene and to gene replacement therapy in retinoblastoma is discussed.