Primary culture of gallbladder epithelial cells obtained from normal rabbits was attempted in collagen gel matrix for up to 6 weeks. Fluid medium containing 0.02% ethylenediaminetetraacetic acid and 0.25% trypsin was poured into the gallbladder lumen. The pellets obtained by centrifugation of recovered fluid contained many isolated epithelial cells and a few small cell clumps. These pellets were dispersed and embedded inside collagen gel matrix and cultured in William's medium E supplemented with fetal calf serum and epidermal growth factor. The three-dimensional outgrowth from individual cells and small cell clumps consisted predominantly of spherical cystic masses 2-4 days later. These cysts contained mucin and were covered by a single layer of cuboidal or low columnar epithelial cells. Electron microscopy revealed the epithelial arrangement of cells lining the cyst walls, and these cells were similar to gallbladder epithelial cells in vivo. These epithelial cells showed active mucin secretion into the cystic cavities. Cytokeratin was diffusely present in the cytoplasm. This isolation and culture system provides a reproducible and consistent method for sustained growth of normal gallbladder epithelial cells from normal tissue in primary culture and seems valuable for investigating pathologic conditions of the gallbladder.