It is estimated that familial aggregation and genetic susceptibility play a role in as many as 10% of pancreatic ductal adenocarcinomas. To investigate the role of germ-line mutations in the etiology of pancreatic cancer, we have analyzed samples from patients with pancreatic cancer enrolled in the NFPTR for mutations in four tumor suppressor candidate genes: (a) MAP2K4; (b) MADH4; (c) ACVR1B; and (d) BRCA2 by direct sequencing of constitutional DNA. These genes are mutated in clinically sporadic pancreatic cancer, but germ-line mutations are either not reported or anecdotal in familial pancreatic cancer. Pancreatic cancer patient samples were selected from kindreds in which three or more family members were affected with pancreatic cancer, at least two of which were first-degree relatives. No mutations were identified in mitogen-activated protein kinase kinase 4 (0 of 22), MADH4 (0 of 22), or ACVR1B (0 of 29), making it unlikely that germ-line mutations in these genes account for a significant number of inherited pancreatic cancers. BRCA2 gene sequencing identified five mutations (5 of 29, 17.2%) that are believed to be deleterious and one point mutation (M192T) unreported previously. Three patients harbored the common 6174delT frameshift mutation, one had the splice site mutation IVS 16-2A > G, and one had the splice site mutation IVS 15-1G > A. Two of the five BRCA2 mutation carriers reported a family history of breast cancer, and none reported a family history of ovarian cancer. These findings confirm the increased risk of pancreatic cancer in individuals with BRCA2 mutations and identify germ-line BRCA2 mutations as the most common inherited genetic alteration yet identified in familial pancreatic cancer.