Epithelial cells, isolated from a normal dog pancreatic duct, were grown on collagen-coated culture inserts suspended above a feeder layer of myofibroblasts. The cells were examined by transmission electron microscopy, immunohistochemistry, cytogenetics, and flow cytometry. In addition, the constitutive and agonist-stimulated mucin secretion of these cells was studied using a [3H]N-acetyl-D-glucosamine labeling assay, and the stimulation of intracellular cAMP was measured. Cells grown on inserts with a feeder layer developed into confluent monolayers consisting of strictly polarized columnar epithelial cells with prominent microvilli, intercellular junctions, and normal chromosomal characteristics. They could be passaged repeatedly without a detectable alteration in their morphology. The cells could also be grown on organotypic cultures, resulting in further differentiated cells simulating in vivo morphology. Immunohistochemistry demonstrated the presence of carbonic anhydrase II in these cells. Cells treated with vasoactive intestinal peptide, epinephrine, and dibutyryl-cAMP demonstrated a marked increase in mucin secretion compared with controls. In parallel experiments, VIP and epinephrine significantly increased intracellular cAMP. In conclusion we have developed a pancreatic epithelial cell preparation with morphology, cytokinetics, chromosomal, and DNA analyses characteristic of normal cells. Similar to normal columnar epithelial cells, these pancreatic duct cells secreted mucin constitutively and responded to agonist by increasing secretion via a cAMP-mediated pathway. They also contained carbonic anhydrase, which indicates that the cells are capable of secreting bicarbonate.