The aim of this study was to examine the role of NO in the control of cardiac metabolism during pregnancy at 60 days of pregnancy (P60) in the dog. There was a basal increase in diastolic coronary blood flow during pregnancy, a statistically significant increase in cardiac output (55±4%) and in cardiac NOx production (44±4 to 59±3 nanomoles/min, p<0.05). Immunohistochemistry of the LV showed an increase in eNOS staining in the endothelial cells at P60. NO-dependent, coronary vasodilation (Bezold-Jarisch reflex) was increased by 20% and blocked by L-NAME. Isotopically labeled substrates were infused to measure oleate, glucose uptake and oxidation. Glucose oxidation was not significantly different in P60 hearts (5.4±0.5 vs. 6.2±0.4 umol/min) but greatly increased in response to L-NAME injection (to 19.9±0.9 umol/min, p<0.05). Free fatty acid (FFA) oxidation was increased in P60 (from 5.3±0.6 to 10.4±0.5 umol/min, p<0.05) and decreased in response to L-NAME (to 4.5±0.5 umol/min, p<0.05). There was an increased oxidation of FFA for ATP production but no change in the respiratory quotient during pregnancy. Genes associated with glucose and glycogen metabolism were downregulated, while genes involved in FFA oxidation were elevated. The increase in endothelium derived NO during pregnancy results in a tonic inhibition of glucose oxidation, and reliance on FFA uptake and oxidation to support ATP synthesis in conjunction with upregulation of FFA metabolic enzymes. Furthermore the acute inhibition of NO shifts the heart away from FFA and towards glucose metabolism despite the downregulation of the carbohydrate oxidative pathway.