Capacity for ATP resynthesis during recovery from ischemia or hypoxia is limited to the size of the adenine nucleotide pool, which is determined in part by the activity of cytosolic 5'-nucleotidase (5'-NT): AMP-->adenosine plus inorganic phosphate (Pi). To define in vivo regulation of 5'-NT, we used the tools of 31P nuclear magnetic resonance (NMR), spectroscopy and chemical assay to measure the substrates (AMP), products (Pi, adenosine, and its catabolites), and inhibitors (Pi and H+) of 5'-NT in isolated perfused rat hearts exposed to hypoxia (where pH remains near 7) and no flow, global ischemia (where pH falls to 6.1). We estimated 5'-NT reaction velocity, assessed the relative contributions of Pi and H+ to enzyme inhibition, and defined the consequences of changes in 5'-NT activity on ATP resynthesis after hypoxia and ischemia. We conclude that (a) 5'-NT is activated during hypoxia and early ischemia but is inhibited during prolonged ischemia, (b) H+ (pH < 6.2) is a potent inhibitor of 5'-NT, and (c) differences in AMP accumulation are sufficient to explain the differences in the capacity for net ATP resynthesis in ischemic and hypoxic tissue. These observations have implications for our understanding of heterogeneity of ischemic injury and myocardial protection during ischemia.