The mechanism by which myocardial blood flow varies in response to metabolic demand is obscure. One hypothesis is a countercurrent regulation of arterial caliber by diffusible substances carried in venous blood. To study the anatomic basis for blood flow regulation, we performed combined arterial and venous injections in 20 human hearts and studied left ventricular intramyocardial vessels with radiography, 100 μm thick sections, and reconstructions of serial histologic sections. Penetrating arteries lie in interstitial spaces and are closely related to accompanying veins. These interstitial veins partially surround and are indented by branch arteries. A second system of veins lies within muscle fascicles between interstitial spaces and is not related to arteries. The isolated veins have collateral connections with interstitial veins and join them in the subepicardium. This vascular anatomy could allow arterial caliber to be regulated by diffusible substances carried in the interstitial veins. Arterial dilatation might prolong this effect by partial obstruction of interstitial veins, with the isolated venous system providing an alternative pathway for venous drainage and washout. The study shows that a vascular arrangement is present in human left ventricular myocardium that could provide a countercurrent regulation of blood flow with diffusible substances carried in venous blood.