Collagenase activity is a critical factor in islet isolation procedures. To evaluate the impact of different collagenase concentrations (Boehringer-Mannheim, Lot 49 and 64) in the canine model of islet isolation, we compared the effect of 500 ml collagenase solution (1.5 mg/ml; Group 1, n=10) with 350 ml of collagenase solution at the same concentration (Group 2, n=4). In both groups, 300 ml were used to distend the pancreas (intra-pancreatic collagenase). Therefore, either 200 or 50 ml of residual solution were added to the chamber (extra-pancreatic collagenase) in Group 1 and 2 respectively. Since approximately 50 ml of solution leak out of the pancreas following distension, at the beginning of the isolation process, the amount of extra-pancreatic collagenase was 250 ml (Group 1) and 100 ml (Group 2). In both groups the system (automated method) was filled with plain HBSS to a final volume of 650 ml resulting in a final extra-pancreatic collagenase concentrations of 0.625 mg/ml in group 1 and 0.25 mg/ml in group 2 respectively. No significant differences were observed in terms of species, age, sex, cold ischemia time and collagenase lot between each group. Islets were isolated by using a modified automated isolation method. The total number of islets/gram tissue, islets EQ/gram tissue and total islets/islets EQ were summarized as following:There is no significant difference in islet yield between the two groups. The data suggests that the critical collagenase concentration seems to be the one used for the distention of the pancreas.