Somatic mutations of the PIK3CA gene, which encodes the p110α catalytic subunit of phosphatidylinositol 3-kinase (PI3K), are frequent in various cancer types. The majority of mutations cluster at hotspots within exons 9 and 20, which encode the helical and kinase domains of p110α. PIK3CA mutations in bile duct and gallbladder carcinomas have not been reported yet. In this study, we analysed 118 carcinomas of the biliary tract and the liver (45 intra- and extrahepatic cholangiocarcinomas (CCA), 23 gallbladder carcinomas, 50 hepatocellular carcinomas) for PIK3CA hotspot mutations using polymerase chain reaction and direct DNA sequencing. PIK3CA missense mutations were found in one of 11 intrahepatic CCA (E545K, 9%), one of 23 gallbladder carcinomas (E542K, 4%), and one of 50 hepatocellular carcinomas (H1047R, 2%). All three mutations represent hotspot mutations, which also occur in other cancer types. PI3K pathway activation in hepato-biliary carcinomas was analyzed using immunohistochemistry for the downstream targets eIF4-E and phosphorylated 4E-BP1 on tissue microarrays. eIF4-E expression was found in 3/13 intrahepatic CCA (23%), 9/38 extrahepatic CCA (24%), 12/34 gallbladder carcinomas (35%), and 9/61 hepatocellular carcinomas (15%). 4E-BP1 phosphorylation was observed in 1/13 intrahepatic CCA (8%), 8/38 extrahepatic CCA (21%), 15/34 gallbladder carcinomas (44%), and 16/61 hepatocellular carcinomas (26%). These results indicate that somatic PIK3CA mutations contribute to the frequent activation of the PI3K/AKT pathway in carcinomas of the biliary tract and liver. © 2008 Wiley-Liss, Inc.