This study evaluated the diagnostic significance of concentrations of the cancer-associated carbohydrate antigen CA 19–9 in pure pancreatic juice (PPJ) collected by endoscopic cannulation. We also attempted to elucidate the features and source of the increased CA 19–9 concentration found in the pancreatic juice of patients with chronic pancreatitis (CP) by means of immunohistochemical staining. The mean output as well as the mean concentration of CA 19–9 in each of the four fractions collected was highest in patients with pancreatic cancer (PC) and also was elevated significantly in patients with CP compared with controls. However, CA 19–9 concentrations were not elevated in patients with cholecystolithiasis. When the cutoff value was set as the mean concentration + 2SD of the controls, significantly elevated concentrations of CA 19–9 were found in the third fraction (secretory phase) in 90% of the patients with PC and 66% of the patients with CP. Immunohistochemical staining revealed that CA 19–9 was expressed more widely in the ductal cells of CP tissues than in those of normal pancreatic (NP) tissues, with CP tissue showing more CA 19–9-positive ductal cells per area than NP tissues. In NP tissue, CA 19–9 was localized to the apical surface and supranuclear regions (apical type) in all the ductal cells stained by the antigen, while ∼50% of cases with CP exhibited a cytoplasmic pattern showing a loss of polarity of the antigen expression. Moreover, this cellular localization pattern was more pronounced in the small ducts that had proliferated and aggregated following the destruction of lobules in CP. These results indicate that although increased concentrations of CA 19–9 in PPJ have no cancer specificity, measurement of CA 19–9 in PPJ can be used as a sensitive marker for some pancreatic disorders, and higher CA 19–9 concentrations in the pancreatic juice of patients with CP may reflect the strong expression of CA 19–9 in the proliferating small ducts associated with CP.