Effects of beta-adrenergic stimulation and atrial pacing on the transmural gradient of intracellular free adenosine were assessed in dog hearts in vivo by measurement of the accumulation of S-adenosylhomocysteine (SAH) in the presence of homocysteine (1.6 mg.kg-1.min-1 iv). Isoproterenol (0.3-0.5 micrograms.kg-1.min-1 iv for 30 min) consistently enhanced left ventricular dP/dtmax (86%), heart rate (38%), and myocardial oxygen consumption (MVO2; 62%) within 3 min while formation and release of adenosine transiently increased. Diastolic aortic pressure fell from 107 +/- 12 to 58 +/- 5 mmHg, and the transmural gradient of SAH was 1.6-, 2.5-, and 4.4-fold increased in subepi-, mid- myo-, and subendocardial layers, respectively. Adenosine formation was inversely related to diastolic aortic pressure. Maintaining aortic pressure greater than 65 mmHg only slightly enhanced venoarterial difference of adenosine despite a greater augmentation of MVO2. Pacing the heart at 209 +/- 4 beats/min enhanced MVO2 by 42% and increased subepi-, midmyo-, and subendocardial levels of SAH by 1.5-, 3.3- and 1.9-fold, respectively. These results demonstrate that in the in situ heart 1) beta-adrenergic stimulation and pacing cause an inhomogeneous transmural increase in free intracellular adenosine mainly affecting the subendocardial and midmyocardial layers; 2) diastolic aortic pressure as the driving force of coronary perfusion is of critical importance for cardiac adenosine formation; and 3) the kinetics of oxygen consumption and adenosine formation are clearly dissociated during beta-stimulation.