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Human telomerase reverse transcriptase (hTERT), one of the subunits of telomerase, is a promising diagnostic marker for pancreatic cancer. In the present study, we did a large-scale analysis of 115 preoperative pancreatic juice specimens to evaluate the feasibility of detection of hTERT expression by immunohistochemistry for preoperative diagnosis of pancreatic malignancy.The expression of hTERT was examined by immunohistochemistry in preoperative pancreatic juice samples.In pancreatic juice samples, hTERT expression was detectable in 84% of pancreatic ductal adenocarcinomas (PDACs), whereas 62% of PDACs were positive by cytology. In intraductal papillary mucinous neoplasms (IPMNs), hTERT expression was detectable in 88% of malignant IPMNs, whereas only 22% were positive by cytology. The sensitivity, specificity, and overall accuracy of hTERT expression for differentiation between carcinoma and other benign diseases were 85.1%, 82.1%, and 84.3%, respectively, whereas the same values for cytologic accuracy were 47.1%, 89.3%, and 57.4%, respectively. When the results of cytology and hTERT expression were combined, the sensitivity and overall accuracy increased to 92.0% and 87.8%, respectively.Our results suggested that the assessment of hTERT expression in preoperative pancreatic juice increased the sensitivity and accuracy of diagnosis of PDACs and malignant IPMNs without using special techniques.