A new antigen associated with pancreatic cancer was prepared by immunoaffinity chromatography using Fab'-Sepharose beads. This antigen was a glycoprotein of large molecular weight (M_r < 8,000,000) in its native state, estimated by size exclusion chromatography on Sephacryl S400. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotting analysis, several cancer-associated glycoconjugates, including CA19–9, CA50, Span-1, Dupan-2, and sialyl SSEA-1, were detected on the antigenic moiety of M_r, 90,000. By an enzyme immunoassay for the antigen, elevated levels were found in pooled sera obtained from patients with various malignant and non-malignant diseases and normal subjects. However, the enhanced expression of CA19–9, Lewis^a, or Lewis^b epitope on the antigen molecule was restricted to the pooled sera from patients with pancreatic cancer. Furthermore, antigens from pancreatic or gastric cancer expressed ligands with intense and specific reactivity for Bauhinia purpurea (BPA), peanut (PNA), and Vicia villosa (VVA) lectins. The present assay system of the antigen, using both monoclonal antibodies (CA19–9, Lewis^a, and Lewis^b) and lectins (BPA, VVA and PNA), will provide a useful approach to the diagnosis of pancreatic cancer.