We have retrospectively evaluated islet isolation records collected from 230 consecutive adult pancreases with 146 pancreases fulfilling all criteria for our evaluation. Fifty-six pancreases procured by our local organ procurement team (33 before in situ vascular flush and 23 after in situ vascular flush with University of Wisconsin [UW] solution) were compared with 90 pancreases received from distant centers that were shipped in UW solution after in situ vascular flushing. Cold storage of 3-26 hr preceded islet isolation using collagenase digestion and Ficoll purification. Recoveries of islets were assessed by duplicate counts of dithizone-stained aliquots before and after purification. Isolations were considered successful if > 100,000 viable islets (islet equivalents to 150 microns) were recovered after purification. Islet function was assessed by in vitro glucose-stimulated perifusion and islets were considered viable if the stimulation index (glucose stimulated over basal insulin secretion) was > 2. Eighty-three percent of the isolations from locally procured, UW-flushed pancreases were successful, as compared with 86% for pancreases that were stored for 3 to 8 hr, 73% for 8 to 16 hr of storage, and 38% for isolations following > 16 hr of cold storage. Increasing the duration of cold storage prior to islet isolation was associated with an increased proportion of failed isolations and decreased measures of islet viability. The actual numbers of islets per gram of pancreas before and after purification were significantly reduced if the hypothermic cold storage was > 16 hr. In vitro viability of isolated islets during glucose-stimulated perifusion showed a higher percentage of viable islets from pancreases with shorter durations of cold storage. For pancreases with > 16 hr of cold storage prior to islet isolation, islet viability was significantly reduced. We conclude that, with the current methods available to recover and store cadaver donor pancreases and the methods currently used to isolate human islets, yields of purified islets decline significantly from human pancreases that have been subjected to cold storage of > 16-18 hr.