We examined the ability of cardiac endothelial nitric oxide synthase (eNOS) to couple myocardial O₂ consumption (MVO₂) and O₂ delivery during pregnancy. Awake dogs were studied using echocardiography before (C), at 40 days (40D), 50 days (50D), 60 days (60D) of pregnancy, and ~14 days post partum (PP). Left ventricular (LV) eNOS, phosphorylated eNOS, and SOD-1 were determined by immunoblotting. MVO₂ of LV tissue samples was measured in vitro in response to increasing doses of bradykinin, enalapril maleate and amlodipine. We examined the changes in passive diameter and flow-dependant arteriolar dilation of coronary arterioles. Echocardiography indicated increases in cardiac output (~60%) during pregnancy. Myocardial eNOS (21±4%), peNOS (19±3%) and SOD-1 (61±2.7%) protein levels were significantly increased at 60D. Bradykinin (BK), enalapril maleate and amlodipine (10^-4 mol/L) decreased MVO₂ in a nitric oxide dependant manner (24 ± 1.3% at C, and 34 ± 2.2% at 60D; 21 ± 1.1% at C, and by 29 ± 1.1 at 60D; 22 ± 2.5% at C, and by 26 ± 1.0% at 60D, respectively). Arterioles from pregnant dogs showed increased flow dependant dilation in response to increased shear stress and larger passive diameter. Nitrite production was stimulated by BK and carbachol in microvessels in vitro, pregnancy enhanced nitrite release. Myocardial eNOS, peNOS, and SOD-1 protein expression are increased during pregnancy, and this increase is associated with enhanced NO dependant control of myocardial oxygen consumption. Thus increases in eNOS and SOD-1 promote the coupling of oxygen delivery and efficiency in the heart during pregnancy.