The role of nucleoside uptake in the enhanced metabolic recovery seen with postischemic ATP.MgCl2 was assessed by determining the effect of S-(p-nitrobenzyl)-6-thioinosine (NBTI) on postischemic ATP recovery in rats given normal saline (NS), ATP.MgCl2, or adenosine after 45 min of bilateral renal ischemia. In NS-infused animals, postischemic administration of NBTI (250 nmol) had no significant effect on the pattern of ATP recovery. In animals given 50 mumol ATP.MgCl2, coinfusion of NBTI significantly reduced the renal ATP content 2 h after reperfusion but blocked only one-half of the enhancement in renal ATP content compared with animals given ATP.MgCl2 alone. In animals postischemically infused with [2,5,8-3H]ATP.MgCl2 (50 mumol) there was significant labeling of nucleotides, nucleosides, and bases after 2 h of reperfusion. The specific activity of the adenosine pool was consistent with significant label uptake in the form of adenosine. Coinfusion of NBTI led to a significant reduction in label incorporation into renal ATP and total adenine nucleotide pools. These data are consistent with an important role for an NBTI-sensitive nucleoside uptake mechanism in the enhanced metabolic and functional recovery observed in ischemically injured kidney treated by postischemic infusion of ATP.MgCl2.