Adenosine, a potent vasodilator, may be involved in the metabolic control of coronary blood flow. However, the site of adenosine formation remains unclear. Evidence has demonstrated that L-homocysteine (L-homo) can decrease the cytosolic adenosine level and α, β-methylene adenosine 5'-diphosphate (AOPCP) can inhibit ect 5'-nucleotidase which catalyzes the formation of adenosine from 5'-AMP at the myocyte membrane. We examined the effects of L-homo and AOPCP on coronary reactive hyperemia following transient coronary occlusion in the open chest dog. Intracoronary L-homo infusion with coronary plasma concentration of 223±54.3↔ M did not affect myocardial reactive hyperemia. Intracoronary AOPCP infusion, which produced coronary plasma concentration of 43.8±20.6 μM and had no effect on hemodynamics or myocardial oxygen consumption, significantly attenuated repayment of flow debt by approximately 30% following coronary occlusions longer than 15 s. Concomitant infusion of L-homo and AOPCP did not change the attenuation of the repayment by AOPCP infusion alone. Percent peak reactive hyperemic response was not affected by L-homo or AOPCP infusion. These results indicated that ect 5'-nucleotidase contributed to adenosine formation during transient ischemia and adenosine is involved in approximately 1/3 of metabolic vasodilation following transient coronary occlusion.