We have modified methods of growing human gallbladder epithelial cells in monolayer and organotypic culture. These cells were grown in the presence of fetal bovine serum and with coculture of feeder layers of human gallbladder fibroblasts. Human gallbladders were obtained from cholecystectomy specimens, and the cells were dissociated with trypsin/EDTA. Cells, which were grown with feeder layer on collagen-coated plates in the presence of 10% FBS, grew rapidly and formed islands of cuboidal cells with morphology typical of epithelial cells in culture, They could be passaged up to four times. The cells were also successfully grown by organotypic technique producing a monolayer of tall, columnar, palisade, epithelial cells. These cells, both in monolayer and in organotypic culture, were positive to antibodies for simple epithelial keratin and negative to antibody for vimentin or any of the mesenchymal antibodies. These cells respond to agonists (prostaglandin E 2. isoproterenol) by the intracellular generation of cAMP. Secreted mucin on the apical surface stained strongly with periodic acid-Schiff. Organotypic culture of human gallbladder epithelium may serve as a cell preparation for the study of pathobiology of columnar epithelial cells.