To study the morphologic and genetic events associated with the carcinogenic process in the pancreas, we have isolated and cultured a cell line of dog pancreatic duct epithelial cells and treated these cells with a carcinogen. The pancreatic duct epithelial cells were plated onto Vitrogen-coated Transwell inserts suspended above a feeder layer of human gallbladder myofibroblasts. The epithelial cells grew steadily into polarized monolayers, could be passaged repeatedly, and demonstrated the typical morphologic, immunohistochemical, and flow cytometric profile of normal well-differentiated columnar pancreatic epithelial cells. After being treated with 10(-5) M N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) for 48 h, the treated cells grew on plastic surfaces. When grown in organotypic culture, the MNNG-treated cells were cuboidal with a multilayered, pseudostratified architecture. Flow cytometry demonstrated aneuploidy and a high percentage of the cells in S phase after reaching confluency, in sharp contrast to untreated cells. Cytogenetic analysis of the MNNG-treated cells revealed frequent chromosomal trisomy and tetrasomy. The secretion of mucin was also different in the MNNG-treated cells versus the untreated cells. The cultured pancreatic epithelial cells may be useful as an assay system to study the genotoxicity of known and potential carcinogens.