Myoglobin may serve a variety of functions in muscular oxygen supply, such as O₂ storage, facilitated O₂ diffusion, and myoglobin-mediated oxidative phosphorylation. We studied the functional consequences of a myoglobin deficiency on cardiac function by producing myoglobin-knockout (myo^−/−) mice. To genetically inactivate the myoglobin gene, exon 2 encoding the heme binding site was deleted in embryonic stem cells via homologous recombination. Myo^−/− mice are viable, fertile, and without any obvious signs of functional limitations. Hemoglobin concentrations were significantly elevated in myo^−/− mice. Cardiac function and energetics were analyzed in isolated perfused hearts under resting conditions and during β-adrenergic stimulation with dobutamine. Myo^−/− hearts showed no alteration in contractile parameters either under basal conditions or after maximal β-adrenergic stimulation (200 nM dobutamine). Tissue levels of ATP, phosphocreatine (³¹P-NMR), and myocardial O₂ consumption were not altered. However, coronary flow {6.4 ± 1.3 ml⋅min⁻¹⋅g⁻¹ [wild-type (WT)] vs. 8.5 ± 2.4 ml⋅min⁻¹⋅g⁻¹ [myo^−/−]} and coronary reserve [17.1 ± 2.1 (WT) vs. 20.8 ± 1.1 (myo^−/−) ml⋅min⁻¹⋅g⁻¹ were significantly elevated in myo^−/− hearts. Histological examination revealed that capillary density also was increased in myo^−/− hearts [3,111 ± 400 mm⁻² (WT) vs. 4,140 ± 140 mm⁻² (Myo^−/−)]. These data demonstrate that disruption of myoglobin results in the activation of multiple compensatory mechanisms that steepen the pO₂ gradient and reduce the diffusion path length for O₂ between capillary and the mitochondria; this suggests that myoglobin normally is important for the delivery of oxygen.