We have documented previously somatic mutations of STK11/LKB11, the gene responsible for Peutz-Jeghers syndrome (PJS), in a small proportion of sporadic pancreatic adenocarcinomas, intraductal papillary mucinous neoplasms (IPMNs), and biliary adenocarcinomas. In this report, we characterize the expression of Stk11, the protein product of the STK11 gene, in a larger series of pancreatic and biliary neoplasms. First, the specificity of the Stk11 antibody was established in 23 neoplasms (22 IPMNs and 1 biliary adenocarcinoma) with known STK11 gene status. Complete absence of labeling was seen in the neoplastic cells of 3 of the 3 (100%) cases with previously documented biallelic inactivation of the STK11 gene, whereas 16 of the 20 (80%) IPMNs, presumably with at least one wild-type STK11 gene, retained Stk11 expression in the neoplastic cells. The marked decrease or absence of Stk11 expression in four neoplasms with wild-type STK11 suggests that additional mechanisms may account for the lack of Stk11 expression. Subsequently, to further evaluate Stk11 expression in pancreatic and biliary neoplasms, tissue microarrays (TMAs) were constructed from a series of nearly 100 ductal adenocarcinomas and biliary neoplasms. Stk11 expression was lost in 4 of the 56 (7%) pancreatic adenocarcinomas and 1 of the 38 (2.6%) biliary cancers by immunohistochemistry; the absence of labeling was confirmed by repeated immunohistochemical labeling of complete tissue sections for the same cases. Thus, Stk11 expression is abrogated in a small proportion of pancreatic and biliary neoplasms. The inactivation of Stk11 in 27% (6/22) of IPMNs versus 7% (4/56) of pancreatic adenocarcinomas suggests genetic disparities in the pathogenesis of these closely related neoplasms. Immunohistochemical analysis for Stk11 expression may be a valid surrogate for genetic analysis of STK11 gene mutations in cancers.