Most studies of mutations in the p53 tumor suppressor gene in tumors have examined only exons 5-8. Our laboratory previously found 64 mutations in exons 5-8 of the p53 gene in 194 primary breast cancers. Herein, we report 18 additional mutations found outside of exons 5-8. Mutations are present in exons 4, 9 and 10, and flanking splice junctions, but not in the promotor region or in exons 1, 2, 3 and 11. No missense mutations are found outside of exons 5-8. Instead, there is a predominance of frameshift mutations with lesser numbers of nonsense and splice site mutations. In contrast, the majority of mutations in exons 5-8 in this sample are missense changes and all of these are at amino acids that are identical in the 11 known p53 sequences that represent about 1.6 billion years of evolutionary divergence. The difference in mutational pattern between these two regions of the p53 gene is due to a lack of missense mutations and inframe microdeletions outside of exons 5-8. A review of our database of p53 mutations (De Vries et al., in preparation) shows that the patterns of mutation inside and outside of exons 5-8 differ in other types of cancers as well. The paucity of missense mutations in exons 2-4 and 9-11 in breast and other cancers (even at amino acids identical throughout p53 gene evolution) suggest that at least some missense mutations result in a phenotype other than malignant transformation. These data also illustrate the importance of examining identical exons when comparing the pattern of p53 gene mutations in different populations.