The transmural distribution of myocardial blood flow across the left ventricles of anesthetized dogs was measured with radioactive microspheres during intracoronary infusion of the endothelium-dependent vasodilators acetylcholine (10 micrograms/min), adenosine triphosphate (ATP; 20 micrograms/min), and arachidonic acid (600 micrograms/min) and the endothelium-independent vasodilator nifedipine (5 micrograms/min). These compounds were administered before and after a 30 min intracoronary infusion of the phospholipase A2 inhibitor quinacrine (300 micrograms/min). Acetylcholine, ATP, and arachidonic acid produced significant (p less than .05) increases in transmural blood flow and in the ratio of subendocardial to subepicardial blood flow (endo/epi) when compared with control. Infusion of quinacrine did not affect this ratio and did not block the increase in transmural blood flow produced by each agent; however, it did block the redistribution of flow to the subendocardium. In contrast, there was no change in endo/epi during intracoronary infusion of nifedipine before and after quinacrine. These results suggest that endothelium-dependent vasodilators produce a preferential increase in subendocardial perfusion via a product of unsaturated fatty acid metabolism.