Using molecular cloning, we earlier isolated the "retinoblastoma gene"; mutations or deletions at this locus are associated with the hereditary predisposition to some human cancers, especially retinoblastoma and osteosarcoma. To develop diagnostic tests for such a predisposition, we identified restriction-fragment-length polymorphisms (RFLPs) within the retinoblastoma gene and tested their usefulness in predicting the risk of cancer in 20 families with members who had hereditary retinoblastoma. We were able to make predictions in 19 of the 20 kindreds. In 18 kindreds, we demonstrated a consistent association of marker RFLPs with the mutation predisposing to retinoblastoma. In the 19th kindred, there may be a lack of cosegregation of the DNA polymorphisms within the gene and the site of the mutation predisposing to retinoblastoma. However, there is uncertainty about the clinical diagnosis of the retinal lesion in a key member of this kindred; if the lesion is not a retinoblastoma, there is no discrepancy between the DNA polymorphisms and the retinoblastoma trait. We conclude that it is feasible and clinically useful to use these DNA polymorphisms to determine the risk of cancer.