BACKGROUND: Optical coherence tomography (OCT) permits high-resolution, real-time, infrared-generated imaging of tissue microstructures by a probe inserted through the endoscope operative channel. Resolution is approximately 10 mu m and the penetration depth of the near-focus probe is about 1 mm. The probe can be inserted into the main pancreatic duct (MPD) through a standard endoscopic retrograde cholangiopancreatography catheter. AIMS AND METHODS: To assess the ability of OCT to identify the structure of the MPD, to distinguish normal and malignant MPD epithelium, and to assess intra- and interobserver reproducibility of OCT images. Multiple sections of neoplastic and non-neoplastic segments of 10 consecutive surgical pancreatic specimens were obtained from patients with pancreatic head adenocarcinoma who had undergone Whipple resection, and repeated OCT radial and longitudinal scanning was done within 1 h of resection and before pathological examination. We compared 249 good-quality images with 100 histopathological sections. RESULTS: OCT recognized a definite, different pattern in 82.9% of tumor-free and in 97.6% of tumor-involved specimens; sensitivity and specificity for discrimination between adenocarcinoma and normal tissue were 78.6% and 88.9%, respectively. Inflammatory and dysplastic changes of the MPD showed an OCT pattern similar to that of the normal tissue in 53.3% of images. Overall, intraobserver reproducibility ranged from 85.1% to 100% and interobserver reproducibility ranged from 69.9% to 100% and from 89.7% to 100% for tumor-free and tumor-involved segments, respectively. CONCLUSIONS: OCT identified the neoplastic and non-neoplastic MPD layer structure and appeared to be a reproducible technique. In non-neoplastic conditions, OCT appeared unable to differentiate between normal and abnormal tissues in about half of the cases.