Recovery of tissue adenosine triphosphate (ATP) levels after transplantation is very important for graft survival. We examined whether the pancreas grafts preserved by the two-layer method have the ability to synthesize ATP after reperfusion, and this is one of the mechanisms of action for the two-layer method in successful pancreas preservation. After preservation by the two-layer method using Euro-Collins' solution (EC) for 48 h (Group 1, n = 5) or simple cold storage in EC for 48 h (Group 2, n = 5), canine pancreas grafts were autotransplanted. In controls (Group 3, n = 5), canine pancreas grafts were autotransplanted without preservation. Graft viability was judged from graft survival after transplantation. Tissue adenine nucleotide concentrations were measured using high performance liquid chromatography after preservation, before reperfusion, and after 1 and 2 h of reperfusion. Graft survival rates were 5 of 5, 0 of 5, and 5 of 5, in Groups 1, 2, and 3, respectively. However ATP tissue levels in Group 1 were significantly higher compared with those in Group 2 after preservation and before reperfusion, respectively (10.95 +/- 1.52 vs. 2.75 +/- 0.33 and 2.90 +/- 0.51 vs. 2.03 +/- 0.68 mumol/g dry weight, p < 0.01 and p < 0.05, respectively). Total tissue adenine nucleotide levels in Group 1 before reperfusion were 7.41 +/- 1.47 mumol/g dry weight, and there was no significant difference compared with Group 2, 6.64 +/- 2.23 mumol/g dry weight. After reperfusion, there was no significant difference of ATP tissue levels between Groups 1 and 2 (4.07 +/- 1.18 vs. 4.48 +/- 1.32, not significant [NS]) after 1 h of reperfusion. However, after 2 h of reperfusion, tissue ATP levels in Group 1 (6.71 +/- 1.19 mumol/g dry weight) were significantly higher than were those in Group 2 (4.51 +/- 0.51 mumol/g dry weight, p < 0.01) and almost at the same levels as control (6.32 +/- 1.62 mumol/g dry weight). It was clear that recovery of ATP after reperfusion did not depend on the residual nucleotides pool but on the ability of the pancreas graft to synthesize ATP after reperfusion. We conclude that oxygenation of the pancreas graft during preservation by the two-layer method allows for ATP synthesis, which is essential in maintaining cellular integrity and leads to maintaining the graft's ability to synthesize ATP promptly after reperfusion. This is one of the mechanisms of action of the two-layer method in successful pancreas preservation.