Myocardial infarction (MI) is caused by coronary atherosclerosis and/or arteriosclerosis. Because endothelial nitric oxide synthase (eNOS) exerts powerful anti-atherosclerotic/anti-arteriosclerotic effects, it is speculated that blockade of eNOS activity might result in MI. However, neither genetic disruption of eNOS nor pharmacologic inhibition of eNOS activity induces MI in animals. On the other hand, intriguingly, genetic disruption of all three nitric oxide synthase (NOS) isoforms (neuronal NOS, inducible NOS, and eNOS) spontaneously caused MI accompanied by multiple cardiovascular risk factors of metabolic origin in mice. This is the first in vivo demonstration showing that the defective NOS system is involved in the pathogenesis of spontaneous MI. Based on the evidence, this review summarizes our current knowledge of spontaneous MI and NOS.