Studies of mutant genotypes of the retinoblastoma susceptibility gene (RB1) in different solid tumors have mainly been concentrated on the demonstration of loss of heterozygosity (LOH) at both internal and external polymorphic sites. One reason for this is the complex organization of the gene. The p105RB protein has been shown to interact with both DNA and regulatory cellular proteins and oncoproteins. The amino acids encoded by exon 21 are implicated in several of these interactions. Both point mutations and intragenic deletions involving exon 21 have previously been reported in human tumors. We have examined RB1 exon 21 from a number of human tumor types where significant LOH in or around the RB1 gene has been reported. DNA from 78 primary tumors was amplified using the polymerase chain reaction (PCR) with primers covering exon 21, followed by constant denaturant gel electrophoresis (CDGE). The 78 tumors included 11 breast carcinomas, 30 nonsmall cell lung carcinomas, 6 colon carcinomas, and 31 sarcomas. The small cell lung cancer cell line NCI-H209, previously shown to harbour a point mutation in codon 706: TGT- greater than TTT (Cys- greater than Phe), was detected using CDGE. Apart from this control mutant cell line, we did not detect any mutations in the examined region in any of the tumors.