The effect of beta-adrenergic blockade with timolol (40 μg/kg) on myocardial blood flow during rest and graded treadmill exercise was assessed in 12 chronically instrumented dogs 10 to 14 days after myocardial infarction was produced by acute left circumflex coronary artery occlusion. During exercise at comparable external work loads, the heart rate-systolic blood pressure product was significantly decreased after timolol, with concomitant reductions of myocardial blood flow in normal, border and central ischemic areas (p < 0.001) and increases in subendocardial/subepicardial blood flow ratios (p < 0.05). In addition to the blunted chronotropic response to exercise, timolol exerted an effect on myocardial blood flow that was not explained by changes in heart rate or blood pressure. At comparable rate-pressure products during exercise, total myocardial blood flow was 24% lower after timolol (p < 0.02) and flow was redistributed from subepicardium to subendocardium in all myocardial regions. Thus, timolol altered myocardial blood flow during exercise by two separate mechanisms: 1) a negative chronotropic effect, and 2) a significant selective reduction of subepicardial perfusion independent of changes in heart rate or blood pressure with transmural redistribution of flow toward the subendocardium.