Description: Myocardial blood flow (MBF) is the critical determinant of cardiac function. However, its response to increases in partial pressure of arterial CO 2 (PaCO 2 ), particularly with respect to adenosine, is not well characterized because of challenges in blood gas control and limited availability of validated approaches to ascertain MBF in vivo. Methods: By prospectively and independently controlling PaCO 2 and combining it with 13 N-ammonia PET measurements, we investigated whether a physiologically tolerable hypercapnic stimulus (~25 mm Hg increase in PaCO 2 ) can increase MBF to that observed with adenosine in 3 groups of canines: without coronary stenosis, subjected to non–flow-limiting coronary stenosis, and after preadministration of caffeine. The extent of effect on MBF due to hypercapnia was compared with adenosine. Results: In the absence of stenosis, mean MBF under hypercapnia was 2.1 ± 0.9 mL/min/g and adenosine was 2.2 ± 1.1 mL/min/g; these were significantly higher than at rest (0.9 ± 0.5 mL/min/g, P 〈 0.05) and were not different from each other ( P = 0.30). Under left-anterior descending coronary stenosis, MBF increased in response to hypercapnia and adenosine ( P 〈 0.05, all territories), but the effect was significantly lower than in the left-anterior descending coronary territory (with hypercapnia and adenosine; both P 〈 0.05). Mean perfusion defect volumes measured with adenosine and hypercapnia were significantly correlated ( R = 0.85) and were not different ( P = 0.12). After preadministration of caffeine, a known inhibitor of adenosine, resting MBF decreased; and hypercapnia increased MBF but not adenosine ( P 〈 0.05). Conclusion: Arterial blood CO 2 tension when increased by 25 mm Hg can induce MBF to the same level as a standard dose of adenosine. Prospectively targeted arterial CO 2 has the capability to evolve as an alternative to current pharmacologic vasodilators used for cardiac stress testing.