Telomerase activation in differentiated cells is associated with cell immortalization. Detection of telomerase expression in cancer provides a potential biomarker to be evaluated in the diagnosis and prognosis of cancer patients. Analysis of telomerase activity in biopsy specimens may serve as a useful marker for the diagnosis of pancreatic adenocarcinoma. This study was conducted to determine if telomerase could be detected in fine needle aspirates from patients with pancreatic carcinoma. The telomeric repeat amplification protocol (TRAP) assay was used to determine telomerase activity from pancreatic cancer cell lines, paired tumor/normal human pancreatic tissues and specimens obtained at the time of diagnostic fine-needle aspiration (FNA) biopsies. In this report, we demonstrate that: i) telomerase activity is present in pathologically confirmed pancreatic cancer specimens, but absent in matched adjacent areas of normal pancreatic tissues; ii) telomerase activity is present in FNA samples obtained at the time of CT-guided FNA of pancreatic adenocarcinomas; and iii) this activity can be detected in material normally discarded during FNA slide preparation. Activation of telomerase can be identified in FNA specimens obtained at the time of routine cytologic diagnosis. These findings may be useful for further diagnostic or therapeutic investigations of telomerase activity in pancreatic carcinoma.