Myoglobin, an intracellular haemoprotein expressed in the heart and oxidative skeletal myofibres of vertebrates, binds molecular oxygen and may facilitate oxygen transport from erythrocytes to mitochondria, thereby maintaining cellular respiration during periods of high physiological demand. Here we show, however, that mice without myoglobin, generated by gene-knockout technology, are fertile and exhibit normal exercise capacity and a normal ventilatory response to low oxygen levels (hypoxia). Heart and soleus muscles from these animals are depigmented, but function normally in standard assays of muscle performance in vitro across a range of work conditions and oxygen availability. These data show that myoglobin is not required to meet the metabolic requirements of pregnancy or exercise in a terrestrial mammal, and raise new questions about oxygen transport and metabolic regulation in working muscles.