Telomerase activity was measured in pancreatic juice obtained by endoscopic retrograde pancreatography from 34 patients (12 with ductal carcinoma, 12 with pancreatic adenoma, and 10 with pancreatitis). The activity in pancreatic juice was expressed as the number of cells of a human pancreatic cancer cell line, MIA PaCa-2, that exhibit an activity equal to that expressed in 1 microg of protein from pancreatic juice. A telomerase ladder was detected in the pancreatic juice obtained from a majority of the patients with ductal adenocarcinoma. The median value of relative telomerase activity in the carcinoma samples was 9.38 (25th percentile, 3.14; 75th percentile, 95.8), a value significantly higher than that derived from patients with either pancreatitis or pancreatic adenoma (P < 0.0001). When a threshold value of relative telomerase activity of 3.00 was used, 75% (9 of 12) of the samples obtained from patients with ductal carcinoma were positive. We conclude that telomerase activity in pancreatic juice differentiates adenocarcinoma from adenoma and pancreatitis and may serve as a useful diagnostic tool.