The transmural distribution of tissue adenosine content was determined in samples of the left ventricle of anesthetized open-chest dogs. Transmural tissue samples were obtained and quickly freeze-clamped during either normal coronary perfusion or partial constriction of the inflow tubing to the left common coronary artery. Inner, middle, and outer segments of the samples were assayed for adenosine, inosine, ATP, creatine phosphate, and lactate. In control experiments, there were no significant differences among the tissue contents of the substances in the three segments. However, during a 50.2 +/- 4.7% reduction of coronary inflow, adenosine content of inner, middle, and outer segments was 0.41 +/- 0.007, 0.030 +/- 0.006, and 0.016 +/- 0.003 nmol/mg protein, respectively. Outer segment content was significantly less than either middle or inner content. Lactate distribution was similar to that of adenosine, whereas ATP and creatine phosphate were lowest in the inner segment. Increases of the inner/outer adenosine ratio occurred when the coronary-ventricular pressure index was lower than 1.2. Thus, selective underperfusion of the subendocardium during reduced coronary inflow can result in greater accumulation of adenosine in that region.