Twenty-one probands, twelve with bilateral and nine with unilateral retinoblastoma, were screened for mutations in the RB1 gene using genomic DNA from peripheral blood leukocytes as well as tumors. Amplification of individual exons and flanking regions of the RB1 gene were carried out, followed by direct sequencing of the amplified products. Sequences of affected individuals were compared with those of controls. Mutations were identified in seven patients, five with bilateral and two with unilateral retinoblastoma. Six out of seven mutations involved the formation of premature termination codons by means of single base substitutions (2), frameshifts due to splice-site mutations (2), or deletion and duplication (2). One missense mutation was identified. Of the remaining fourteen patients, seven with bilateral disease had no mutations in peripheral blood (7 cases) or tumors (3/7 cases). Analysis of the peripheral blood of seven patients with unilateral disease also showed no mutations. Mutations were detected in about one-third of the cases, suggesting that hemizygous deletions at the RB1 locus or mutations outside the coding regions of RB1 may be responsible for the disease in the remaining patients.