There is uncertainty about which metabolic pathway is most important for controlling myocardial cytosolic adenosine concentrations in the normoxic heart: AMP hydrolysis, linked to energetic status, or energy independent transmethylation (via S-adenosylhomocysteine hydrolysis) To answer this question, intracellular cycling of AMP and adenosine was abolished using an intracoronary infusion of the adenosine kinase blocker iodotubercidin (ITC), in the presence of adenosine deaminase inhibition. ITC caused a 10-fold increase in the coronary venous release rate of adenosine to 3.4 + 0.3 (mean + SE, n=5) nmol min-1g-1, representing total normoxic myocardial adenosine production. To determine the relative roles of AMP hydrolysis and transmethylation, parallel experiments tested the effect of ITC during blockade of transmethylation using adenosine dialdehyde. In these experiments, venous adenosine release increased to similar levels of 3.4 + 0.5 (n=6) nmol min-1g-1. The possibility that ITC caused increased adenosine release by interfering with myocardial energetics was ruled out in separate 31P NMR experiments. Mathematical modeling analysis of the adenosine results indicated that AMP-adenosine cycling causes increased sensitivity of cytosolic adenosine concentrations to increases in the rate of AMP hydrolysis. It is concluded that 1) at least 90% of the adenosine produced intracellularly is normally rephosphorylated to AMP without escaping into the venous effluent, 2) AMP hydrolysis is the dominant pathway for normoxic adenosine production, and 3) AMP-adenosine cycling serves to amplify the relative importance of energy dependent AMP hydrolysis over that of transmethylation in controlling cytosolic adenosine concentrations.