Methylthioadenosine phosphorylase (MTAP) plays an important role in the salvage pathway for the synthesis of adenosine. Novel chemotherapeutic strategies exploiting the selective loss of MTAP function in cancers have been proposed. The MTAP gene, on chromosome 9p21, is frequently included within homozygous deletions of the p16INK4A/ CDKN2A gene. Biallelic deletions of the p16INK4A/CDKN2A gene are found in 40% of pancreatic cancers, suggesting that the MTAP gene may be frequently inactivated in pancreatic cancer and that selected patients with pancreatic cancer may benefit from therapies targeting this loss. We immunolabeled six xenografted pancreatic cancers with known MTAP and p16INK4A/CDKN2A gene status and found that immunolabeling mirrored gene status. Loss of expression of both MTAP and p16 was observed only in those pancreatic cancers with homozygous deletions that encompassed both the MTAP and p16INK4A/CDKN2A genes. We then immunolabeled a series of 320 microarrayed infiltrating pancreatic adenocarcinomas, 35 biliary adenocarcinomas, 54 ampullary cancers, and 35 noninvasive intraductal papillary mucinous neoplasms. Immunolabeling for MTAP was lost in 91 of the 300 (30%) evaluable pancreatic cancers, 9 of 54 (17%) ampullary cancers, 4 of 33 (12%) biliary cancers, and in 1 of 35 (3%) IPMNs. All neoplasms with loss of MTAP labeling also demonstrated loss of p16 labeling. These results suggest that MTAP expression is lost in approximately 30% of infiltrating pancreatic cancers and in a lower percentage of other periampullary neoplasms, that this loss is the result of homozygous deletions encompassing both the MTAP and p16INK4A/CDKN2A genes. Thus, pancreatic cancer is a promising cancer type in which to explore novel chemotherapeutic strategies to exploit the selective loss of MTAP function.