Fourteen fetal lambs were instrumented with atrial, coronary sinus, and arterial catheters and a proximal left circumflex coronary artery Doppler probe and were studied at a mean gestational age of 130 +/- 3 (SD) days, 7 +/- 2 days after surgery. Myocardial blood flow was assessed using 15-microns microspheres and Doppler flow velocities. In 11 fetuses, the maximal myocardial flow response to left atrial adenosine infusion was 802 +/- 215 ml.min-1 x 100 g-1, 3.5-fold greater than baseline flow. Acute fetal hypoxemia in six fetuses to an arterial PO2 of 8.8 +/- 0.8 mmHg and an arterial O2 content (CaO2) of 1.7 +/- 0.2 ml/dl was not associated with significant change in coronary perfusion pressure; yet left ventricular myocardial flow increased to 1,020 +/- 198 ml.min-1 x 100 g-1, a value significantly greater than that seen with adenosine (P < 0.05). Left atrial N omega-nitro-L-arginine (L-NNA), a competitive inhibitor of nitric oxide synthase (NOS), was infused at a dosage of approximately 1 mg.kg-1.min-1 for 60 min in 10 fetuses. Although L-NNA was associated with a significant increase in arterial pressure, left ventricular myocardial flow decreased (162 +/- 79 ml.min-1 x 100 g-1) as did myocardial O2 consumption (P < 0.05). Acute hypoxemia in five fetuses that received L-NNA was associated with significant further increases in systemic arterial pressure; however, left ventricular myocardial flow was only 771 +/- 237 ml.min-1 x 100 g-1, a value similar to that seen with adenosine and approximately 75% of that seen with acute hypoxemia alone. We conclude that nitric oxide plays an important role in the regulation of fetal myocardial flow during basal conditions as well as in the exuberant vasodilatory response associated with acute hypoxemic stress.