To test the diagnostic utility of pancreatic digestive enzyme immunohistochemistry in liver cancers, the expression of three pancreatic digestive enzymes (trypsinogen, chymotrypsinogen and pancreatic lipase) was investigated in cholangiocarcinoma (CC) (n = 42), hepatocellular carcinoma (HCC) (n = 35), combined HCC-CC (n = 11) and metastatic adenocarcinoma (MA) of the liver (n = 34; 4 gastric cancer, 5 pancreatic cancer and 25 colon cancer). In CC, 15 (36%) expressed one or more of these enzymes, while the remaining 27 (64%) did not express any enzymes. In MA, 13 (38%) expressed one or more of these enzymes, while the remaining 21 (62%) did not express any enzymes. Expression of trypsinogen, chymotrypsinogen and lipase was noted in 15 CC (36%), 11 CC (25%) and 15 CC (36%), respectively, and in 9 MA (26%), 6 MA (18%) and 13 MA (38%), respectively. There was no significant difference in the positive ratio of each enzyme between CC and MA. In positive cases, the enzymes were expressed with a cytoplasmic granular pattern. In MA, there was no significant difference in the positive ratio of the enzymes among the primary sites. In contrast to CC and MA, these enzymes were not expressed in any cases of HCC and combined HCC-CC. These data suggest that pancreatic digestive enzyme immunohistochemistry may be useful for differential diagnosis between HCC and CC or MA as well as between combined HCC-CC and CC or MA, but it is not useful for differential diagnosis between CC and MA. A positive reaction for these enzymes is indicative of CC or MA and is against the diagnosis of HCC or combined HCC-CC, and a negative reaction is noncontributory to the differential diagnosis.