Patients who initially develop an upper aerodigestive tract cancer have an increased risk of second primary cancers. We examined TP53 and RAS mutations and p53 protein in 21 tumors from 10 patients with upper aerodigestive tract cancer who developed a metachronous tumor, to assess the genetic changes that occur in multiple primary tumors from the same individual. Thirteen of 21 (62%) tumors were found to have mis-sense mutations of either TP53 or RAS. Six tumors had TP53 mutations in codons 5 to 8 and 10 tumors from 7 patients had mutations of codons 12 or 13 of K-RAS. Only one patient had concordance of a mutation in 2 tumors; this mutation occurred in K-RAS and was accompanied by discordance of TP53 mutation. Three patients had tumors discordant for both TP53 and RAS mutations. Smoking-related tumors had TP53 and RAS mutations which were transversions in 11 (9 G:C to T:A and 2 G:C to C:G) and transitions in 3 (2 G:C to A:T and 1 A:T to G:C). Tumors not associated with smoking contained only transitions (both G:C to A:T). p53 protein was detected by immunohistochemistry in 7 of 13 (54%) tumors and was concordant in the multiple tumors of 3 patients. Three of the 7 tumors staining for p53 also had TP53 mutations. Thus, genetic alterations are discordant in multiple primary cancers and the pattern of mutations is similar to that found in patients with a single primary tumor, supporting the concept that these cancers arise independently.