The transcription of a eukaryotic gene is a consequence of intricate interactions between members of a set of transcription factors. We describe here evidence indicating that at least two distinct DNA-binding factors play an important part in the transcription of the human retinoblastoma gene (Rb). One of the factors reacts with a sequence overlapping with a potential Sp1 recognition sequence in the promoter region of the gene, the other with a nearby ATF recognition sequence. We have identified two naturally occurring point mutations in these recognition sequences that cause hereditary retinoblastoma. The nuclear factors do not bind to the mutant sequences. We infer that these nuclear factors are necessary for the expression of the Rb gene and the suppression of cancer.