We have demonstrated that a high adenosine triphosphate (ATP) level in a canine pancreas during preservation by the two-layer method is an important determinant for the ultimate success of pancreatic transplantation. In this study, we investigated (a) the effect of factors that seemed to have an influence on energy metabolism in the canine pancreas at the tissue ATP level and (b) graft viability during preservation by the two-layer method. ATP tissue concentration was determined by high-performance liquid chromatography and graft viability was assessed on the basis of survival rate following autotransplantation. First, the pancreas was harvested from either 72-h-fasted (n = 5) or fed dogs (n = 5) and preserved by the two-layer Euro-Collins solution (EC)/perfluorochemical (PFC) method for 24 h. All the pancreatic grafts were viable in both fed and fasted groups. There was also no significant difference in ATP tissue concentration between the two groups (7.48 +/- 0.55 vs. 7.03 +/- 0.74 micromol/g dry weight, NS). Second, the pancreatic grafts subjected to 60 min of warm ischemia were preserved by either the two-layer (EC/PFC) or (EC + adenosine/PFC) method for 24 h. Without adenosine, ATP tissue concentration did not recover (1.62 +/- 0.26 after warm ischemia vs. 1.56 +/- 0.40 micromol/g dry weight after preservation, NS) and all the pancreatic grafts failed. However, provision of adenosine led to restoration of ATP tissue levels (1.90 +/- 0.53 vs. 7.23 +/- 2.17 micromol/g dry weight, P < 0.01) and four of five grafts functioned immediately and maintained normoglycemia after transplantation. These results clearly demonstrated that the nutritional state of the pancreatic graft before procurement had no influence on ATP tissue level as well as graft viability during 24-h preservation by the two-layer method. On the other hand, provision of adenosine during 24-h preservation enhanced ATP synthesis of the pancreatic tissue, thereby improving viability of the ischemically damaged pancreas.