To elucidate the physiological role of the AMP-adenosine metabolic cycle and to investigate the relation between AMP and adenosine formation, the O₂ supply of isolated guinea pig hearts was varied (95% to 10% O₂). The net adenosine formation rate (AMP→adenosine) and coronary venous effluent adenosine release rate were measured; free cytosolic AMP was determined by ³¹P-nuclear magnetic resonance. Switching from 95% to 40% O₂ increased free AMP and adenosine formation 4-fold, whereas free cytosolic adenosine and venous adenosine release rose 15- to 20-fold. In the AMP range from 200 to 3000 nmol/L, there was a linear correlation between free AMP and adenosine formation (R²=.71); however, adenosine release increased several-fold more than formation. At 95% O₂, only 6% of the adenosine formed was released; however, this fraction increased to 22% at 40% O₂, demonstrating reduced adenosine salvage. Selective blockade of adenosine deaminase and adenosine kinase indicated that flux through adenosine kinase decreased from 85% to 35% of adenosine formation in hypoxia. Mathematical model analysis indicated that this apparent decrease in enzyme activity was not due to saturation but to the inhibition of adenosine kinase activity to 6% of the basal levels. The data show (1) that adenosine formation is proportional to the AMP substrate concentration and (2) that hypoxia decreases adenosine kinase activity, thereby shunting myocardial adenosine from the salvage pathway to venous release. In conclusion, because of the normal high turnover of the AMP-adenosine metabolic cycle, hypoxia-induced inhibition of adenosine kinase causes the amplification of small changes in free AMP into a major rise in adenosine. This mechanism plays an important role in the high sensitivity of the cardiac adenosine system to impaired oxygenation.