Little is known about the vascular and metabolic adaptations that take place in the fetal heart to maintain cardiac function in response to increased load. Chronic fetal anemia has previously been shown to result in increased ventricular mass, increased myocardial vascularization, and increased myocardial expression of hypoxia-inducible factor-1 (HIF-1) and vascular endothelial growth factor (VEGF). We therefore sought to determine if chronic fetal anemia induces expression of HIF-1 regulated angiogenic factors and glycolytic enzymes in the fetal myocardium. Anemia was produced in chronically instrumented fetal sheep by daily isovolemic hemorrhage (80-100 ml) for either 3 d (n=4) or 7 d (n=11) beginning at 134 days gestation (term 145 days). Catheterized, non-bled twins served as controls. Isovolemic hemorrhage over 7 d resulted in decreased fetal hematocrit (37±1 to 20±1%) and arterial oxygen content (6.5±0.4 to 2.8±0.2 ml O₂/dL). Myocardial blood flow and vascularization were significantly increased after 7 d of anemia. Myocardial HIF-1 protein expression and VEGF (LV), VEGFR-1 (RV) and VEGFR-2 (RV, LV) mRNA levels were elevated (p< 0.05) in 7 d anemic compared to control animals. Myocardial expression of the glycolytic enzymes aldolase, lactate dehydrogenase A, phosphofructokinase-liver and phosphoglycerol kinase were also significantly elevated after 7 d of anemia. Despite the absence of a significant increase in myocardial HIF-1 protein in 3 d anemic fetuses, expression of VEGF, VEGFR-1 and the glycolytic enzymes was greater in 3 d compared to 7 d anemic animals. These data suggest that HIF-1 likely participates in the fetal myocardial response to anemia by coordinating an increase in the expression of genes that promote capillary growth and anaerobic metabolism. However, factors other than HIF-1 also appear important in the regulation of these genes. We speculate the return of mRNA levels of angiogenic and glycolytic enzymes toward control levels in the 7 d anemic fetus is explained by a significantly increased resting myocardial blood flow, resulting from coronary vascular growth and increased coronary conductance, and a return to a state of adequate oxygen and nutrient delivery, obviating the need for enhanced transcription of genes encoding angiogenic and glycolytic enzymes.