Epimorphin was recently described as a mesenchymal factor modulating morphogenesis of murine mammary ducts, skin, liver, and lung in vitro. In this study epimorphin was analyzed in a human, pancreatic adenocarcinoma cell line (A818-6) which develops single layer epithelial hollow spheres resembling normal pancreatic ductal structures in vitro. Soluble 34- and 31-kD isoforms of epimorphin were found in the culture supernatant of A818-6 cells. In lysates of A818-6 cells we detected the 34-and 31-kD isoforms and the dimers, and in lysates of fibroblasts the 150-kD tetramers of epimorphin additionally. A neutralizing monoclonal antibody against epimorphin (MC-1) efficiently blocked the development of hollow sphere structures from A818-6 cells. Coculture of A818-6 cells with fibroblasts stimulated the development of hollow sphere structures in general and increased differentiation in 5–6-d-old hollow spheres. A818-6 hollow sphere development in the presence of fibroblasts was also blocked by MC-1. In this novel system for human duct-like differentiation of pancreatic epithelial cells, we provide evidence for an autocrine and paracrine function of epimorphin as a major mediator for morphogenesis.