With a limiting dilution technique, clones have been established from the human pancreatic adenocarcinoma cell line, HPAF. Phenotypic analysis by a panel of murine monoclonal antibodies demonstrated distinct profiles of antigenic expression between the clones. However, identical isozyme patterns of different clones indicated their common origin from the parental HPAF cells. Two clones, CD11 and CD18, appeared to be arrested at different stages of secretory epithelial cell differentiation. CD11 cells demonstrated many characteristics of a well-differentiated state, including the formation of ductal structures with polarized, long columnar-shaped cells, the presence of secretory granules in the cytoplasm, high DU-PAN-2 antigen expression in nude mouse xenografts, and a longer doubling time (42 h) in tissue culture. In contrast, CD18 cells exhibited characteristics of a poorly differentiated state, including solid nests of isoprismatic cells without luminal spaces and cellular polarization, absence of secretory granules and DU-PAN-2 antigen expression in xenografts, and a shorter doubling time (26 h) in tissue culture. Since no culture systems of normal pancreatic ductal cells are currently available, these two pancreatic adenocarcinoma clones may provide a unique system to study genes and antigens related to pancreatic ductal cell differentiation.