DNA from 61 unrelated patients with adenomatous polyposis coli (APC) was examined for mutations in three genes@ Pi, SRP79, and DP2. 5) located within a 166 kb region deleted in two of the patients. The intron-exon boundary sequences were defined for each of these genes, and single-strand conformation polymorphism analysis of exons from DP2. 5 identified four mutations specific to APC patients. Each of two aberrant alleles contained a base substitution changing an amino acid to a stop codon in the predicted peptide; the other mutations were small deletions leading to frameshifts. Analysts of DNA from parents of one of these patients showed that his 2 bp deletion is a new mutation; furthermore, the mutation was transmitted to two of his children. These data have established that DP2. 5 is the APC gene.