A series of 12 cell fusions of BALB/c mice spleen cells with the SP2/O-Ag14 mouse myeloma cell line were performed after immunizations with normal human pancreas to generate monoclonal antibodies (mAbs) with high affinity against antigens of the exocrine pancreas. The immunohistologic tissue screening resulted in the selection of 14 clones that were further characterized on frozen tissues, tumor cell lines, and lectin binding assays. Four mAbs reacting with pancreatic acini detected granular antigens and three mAbs reacted with acinar cell membrane antigens. Six other clones reacted with pancreatic ducts; among these were three clones positive with antigens in both acini and ducts. Among the clones reactive with secretory products, there was one pair of mAbs identified as pancreas specific (P78C9/P79D4); the other secretory antigens (identified by P97F3 and P109H1) were also detectable in other exocrine organs, but were absent from the gastrointestinal mucosa. Two clones reactive with acinar cell membranes (P96H2 and P100H1) were also positive with hepatocytes. All but four antigens were detectable in pancreatic cancer cell lines Capan-1, Capan-2, and DAN-G. On the whole, immunizations were more effective using whole pancreatic tissues (eight clones selected) compared with single-cell suspensions as immunogen (four clones) or cell membrane preparations (two clones). Therefore, the immunization and screening strategies used in this study resulted in the generation of new mAbs against specific substructures of the exocrine pancreas. The shared expression of some of these antigens with other exocrine organs and pancreatic adenocarcinoma cell lines suggests that these mAbs detect antigens that are characteristic for the exocrine system and could therefore be useful for the study of the exocrine pancreas and pancreatic adenocarcinomas.