Adenosine levels present in the interstitial fluid and coronary effluent of the aged heart exceed those of the young adult heart. The present study investigated mechanisms in the Fischer 344 rat heart which may be responsible for the observed differences. (1) Total production of adenosine was determined in isolated perfused hearts by measuring coronary effluent adenosine content while inhibiting adenosine deamination and rephosphorylation with erythrohydroxy-nonyladenosine (EHNA) and iodotubercidin (ITC), respectively. Total adenosine production was similar in both young (3-4 month) and aged (20-21 month) hearts at 31.8 +/- 6.6 and 38.4 +/- 3.3 nmol/min/g dry wt, respectively. However, stimulation with the beta-adrenergic agent, isoproterenol, elicited a significantly greater increase in adenosine production in the young vs. aged heart. (2) Adenosine transport was evaluated in isolated perfused hearts by determining 14C uptake by the myocardium after 20 min of 14C-adenosine perfusion. Adenosine uptake in the agent-free heart was found to be decreased 17 to 25% in aged compared to young adult hearts. (3) Adenosine transport characteristics were determined with nitrobenzylthioinosine saturation-binding studies in ventricular membrane preparations. The Bmax values were significantly lower in aged than young adult hearts (140.2 +/- 1.5 fmol/mg and 191.9 +/- 2.3 fmol/mg in aged and young hearts, respectively) indicating a decreased number of transporter sites in the aged heart. However, the values for Kd were decreased with aging, suggesting an increase in the affinity of the transporter for adenosine in the aged vs. young adult heart. (4) The activities and kinetics of adenosine kinase were determined in homogenates of aged and young adult ventricular myocardium. No statistical difference was found between the two activities. Taken together these results suggest that increased interstitial adenosine levels in the aged heart result from decreased uptake of adenosine by the ventricular myocardium.