Rat pancreatic acinar cells have been dissociated and maintained in culture under specific conditions which allow the retention of their differentiated state and three-dimensional organization. When cultured on a basal lamina (basement membrane) matrix, the cells first formed large monolayer patches and then reorganized themselves into acini-like structures. The cells regained their polarity around luminal spaces which appeared to be sealed off by well developed junctional complexes. Typical microvilli appeared at the "apical" plasma membrane projecting themselves into the luminal spaces. The intracellular organization resembled that of the cells in situ: a well developed rough endoplasmic reticulum located towards the "base" of the cell around a nucleus; a supranuclearly positioned Golgi apparatus and numerous secretory granules located in the "apical" region of the cell. Immunocytochemistry has revealed the presence of two pancreatic enzymes, amylase and chymotrypsinogen, in the various cellular compartments involved in secretion; the rough endoplasmic reticulum and Golgi cisternae as well as in the secretory granules. Biochemical evaluations have also shown the presence of amylase in the acinar cells and culture medium. These results thus demonstrate that dissociated pancreatic acinar cells maintained in culture under specific conditions reaggregate themselves into acini-like structures and retain their differentiated morphology as well as their ability to secrete.