We previously reported 9 unilateral, sporadic retinoblastomas with hypermethylation in the 5' region of the RB gene, and we found that CpG methylation in the RB promoter inhibits the binding of the retinoblastoma binding factor 1 (RBF-1) and the activating transcription factor (ATF)-like factors, thereby resulting in a considerable reduction in RB promoter activity. In this study, we screened for hypermethylation in 121 additional cases of retinoblastoma, and found 5 tumors with hypermethylation, including 4 unilateral, sporadic tumors, and one hereditary tumor. The hereditary tumor had a germline deletion of one allele, and the hypermethylation was an acquired, epigenetic change in the other allele. Another tumor had hypermethylation restricted to approximately 800 base pairs in the RB promoter region including the essential RBF-1 and ATF sites. The frequency of hypermethylation in unilateral, sporadic tumors was 9.3% combining our previous and present examinations (13 among 140), whereas the frequency was 1.0% in bilateral hereditary tumors (one among 101). The statistical analyses using the chi-square test indicated significant correlation between hypermethylation and unilateral, sporadic tumors (p < 0.05). These results suggest that hypermethylation in the RB gene is always an acquired, epigenetic change and causes about 9% of unilateral, sporadic tumors.