Submandibular glands of 4-week-old rats were dissociated by a procedure involving digestions with collagenase and hyaluronidase, chelation with ethylenediaminetetraacetic acid, and mechanical force. The isolated cells were purified by centrifugation in a Ficoli solution and were maintained in culture for 36 hours. On the basis of trypan blue exclusions, about 70 per cent of the dissociated cells were viable. Electron microscopic observations indicated that the isolated acinar cells and intercalated and striated duct cells retained their essential in situ ultrastructural characteristics. During a 36-hour culture period the number of viable cells declined to about 40 per cent, and the various cell types formed mixed aggregates. The ultrastructural features of the intercalated and duct cells changed relatively little, but the acinar cells revealed several structural alterations. These included a decrease in the number of the secretory granules, fusions of the secretory granules, and an increase in the rough surfaced endoplasmic reticulum. In general, the polarity of acinar cells became less distinct. The endogenous peroxidase activity in the acinar cells gradually diminished during the culture. Isoproterenol when added to the cultured cells failed to stimulate the incorporation of radioactive thymidine or the discharge of the secretory material from the acinar cells.