We describe a method for the preparation of viable islets from the pancreas of normal 8-week-old female Syrian golden hamster, based on the injection of the pancreatic duct with collagenase and on mechanical dissociation which liberates islets that maintain their normal morphological appearance and physiologic function. In a series of 11 animals, we examined the dose response of intraductal collagenase on islet yield. The mean number of isolated islets was 423 with a range from 130 to 873 per pancreas. Islet yield was most dependent on the concentration of collagenase solution used to inject the duct. The optimal concentration was determined to be 3.5 mg/ml when a total volume of 3.0 ml was injected. Islets responded to glucose stimulus in a normal biphasic pattern. Mesh filtration, rather than Ficoll, can be performed rapidly and results in a high yield of functional islets with minimal contamination by acinar tissue.