Methods have been developed for culturing a dividing population of morphologically differentiated rat parotid, lacrimal, and pancreatic acinar cells in vitro. Isolated acinar cells were plated onto tissue culture dishes coated with a three-dimensional, reconstituted basement membrane gel. After attachment in Ham's nutrient mixture F12, the cells were cultured at 35 degrees C in F12 supplemented with 10% heat inactivated rat serum, epidermal growth factor, dexamethasone, insulin, transferrin, selenium, putrescine, reduced glutathione, ascorbate, penicillin, streptomycin, and the appropriate secretagogue. Under these conditions, the cells attached rapidly and DNA synthesis was initiated within 2 to 3 d. Although the cells flattened on the substratum, they continued to maintain their differentiated morphology. The cells contained secretory granules, and the secretory enzymes peroxidase and amylase could be detected. The use of a reconstituted basement membrane gel proved critical for the attachment and growth of exocrine acinar cells.