We have demonstrated that a two-layer (University of Wisconsin solution [UW]/perfluorochemical [PFC]) cold storage method restores the function of ischemically damaged pancreas during 24-hr preservation in canine autotransplantation model. The purpose of this study was to examine the possibility of a long-term preservation of the ischemically damaged pancreas by the two-layer (UW/PFC) method. After 60 or 90 min of warm ischemic time, pancreas grafts were preserved by the two-layer (UW/PFC) method or a simple cold storage in UW alone for up to 96 hr. A K value of i.v. glucose tolerance test more than 1.0 2 weeks after autotransplantation was considered successful preservation. After 60 min warm ischemia, limitation of preservation time by the simple cold storage in UW was 24 hr (5/5 100% and 0/5 0%; 24- and 48-hr preservation, respectively). However, the two-layer method made it possible to extend the preservation time up to 48 hr (5/5 100%, 5/5 100%, 2/5 40%, and 0/5 0%; 24-, 48-, 72-, and 96-hr preservation, respectively). After 90 min warm ischemia, the simple cold storage in UW was not effective even for 24-hr preservation (0/5 0%). However, 48-hr preservation was successful by the two-layer (UW/PFC) method (5/5 100%, 5/5 100%, and 0/5 0%; 24-, 48-, and 72-hr preservation, respectively). After preservation by the two-layer (Um/PFC) method, ATP tissue concentrations of viable grafts were significantly higher compared with nonviable grafts (9.11+/-3.05 (n=22) versus 5.22+/-1.02 (n=13) mu mol/g dry wt, P<0.001). Based on analysis of individual ATP for each graft, if an ATP concentration of 6.0 mu mol/g dry weight was determined as a critical value for doing the transplant, sensitivity, specificity, positive predictive value, and negative predictive value were 100%, 84.6%, 91.7%, and 94.3%, respectively. This study clearly demonstrated that 48-hr preservation of the canine pancreas subjected to either 60 or 90 min warm ischemia was successfully achieved by the two-layer (UW/PFC) cold storage method, and ATP tissue concentration at the end of preservation by this method would predict the posttransplant outcome of the ischemically damaged pancreas just prior to transplantation.