We analyzed the preexisting donor factors and isolation variables that affected isolation of human islets of Langerhans. Sixty-nine pancreata from cadaveric donors were analyzed for donor factors of age, gender, body mass index, cause of death as well as graft factors of cold ischemia time, pancreas status, distensibility during intraductal collagenase distension and time of collagenase expansion and digestion. Islet isolations that recovered >100,000 IEQ (n = 53) were compared to those generating less than 100,000 IEQ (n = 16) to analyze the factors affecting islet yield during donor harvest and isolation procedures. The mean islet recovery was 216.0 x 10(3) (IEQ) or 2840 (IEQ) per gram of pancreas. Mean purity was 54%. The success rate of islet isolation was 76%. Mean age was 31 years, and mean cold ischemia time was 6.9 hours. In univariate analysis, the status of the pancreas was the only significant factor for successful isolation, and gender, time of collagenase expansion and digestion were marginal factors. In stepwise multivariate logistic regression analysis of donor and isolation-related factors, donor gender, pancreas status and digestion time were significant factors. During the same period we performed three cases of clinical islet allotransplantation and one autotransplantation. This study confirmed that the same donor factors and variables in the isolation process can affect the ability to obtain successful human islet isolation. Enough experience and pertinent review of donor and isolation factors can make islet isolation consistent, supporting clinical islet transplantation without unnecessary cost.