Summary The association between Ki-ras mutations and proliferation activity was investigated in a comprehensive series of biliary tract carcinomas (BTCs). We precisely microdissected samples of tissue from paraffin-embedded sections of 77 BTCs including 22 intrahepatic cholangiocarcinomas (ICCs), 36 extrahepatic cholangiocarcinomas (ECCs), and 19 gall bladder carcinomas (GBCs). Ki-ras mutations at exons 1 and 2 were determined by the polymerase chain reaction -single strand conformation polymorphism (PCR-SSCP) method and confirmed by direct sequencing. Proliferation activity was immunohistochemically assessed to generate proliferating cell nuclear antigen labelling indices (PCNA LIs). Ki-ras mutations were detected in 10 of 22 ICCs (45%), 24 of 36 ECCs (67%), and in 16 of 19 GBCs (84%). The frequency of Ki-ras mutations in peripheral type ICCs was 33% (4 of 12) and that in the hilar type ICCs was 60% (6 of 10). In ECCs the highest value of 82% (9 of 11) was found for carcinomas located in the lower part of the biliary tree. Mean PCNA LI in mutation-positive BTCs was significantly elevated compared with the mutation-negative value. These results indicate frequent involvement of Ki-ras mutations in BTCs, especially in GBCs and in distal ECCs, and that carcinomas harbouring a mutation feature high cell proliferation activity.