Morphologically and functionally intact acinar cells have been obtained from the rat parotid gland through enzymatic dispersion with pure collagenase, hyaluronidase, and trypsin as well as mild mechanical forces. Cell yields of 30-50% of the original tissue weight with over 95% acinar cells were accomplished. The cells in suspension assumed a more or less spherical shape but the intracellular polarity of organelle distribution was maintained. The cells in suspension at 37 degrees C maintained stable monovalent cationic composition but lost potassium and gained sodium rapidly upon exposure to ouabain, 10(-5) M. The intracellular amylase concentration and the patterns of secretion of amylase and of synthesis of cyclic AMP by the cells in response to adrenergic stimulation with epinephrine or isoproterenol were comparable to those of the intact gland in situ. In addition, the cells showed good O2 consumption and maintained it constant for periods up to 8 h. These cells could be used as experimental tools for in vitro studies of receptor physiology and biochemistry, cell membrane function, cellular secretory mechanisms, and other parameters of exocrine gland cell physiology.