Myocarditis is an important cause of heart failure in adolescents and young adults and is caused, most commonly, by viral infections. Viral myocarditis is characterized by cardiac inflammation and cardiomyocyte necrosis. The molecular pathogenesis of viral myocarditis is incomplete and specific therapies are not available. Proinflammatory cytokines such as IL-1beta, TNF-alpha and IL-6 have been implicated in the pathogenesis of myocarditis caused by encephalomyocarditis virus (EMCV) infection, a model of viral myocarditis in mice. Substance P (SP), a neuropeptide and pain transmitter, stimulates the production of proinflammatory cytokines and has been demonstrated by us and others to contribute to the pathogenesis of several viral, protozoan and helminth infections in mouse and man. Receptors for SP are expressed on the surface of cardiomyocytes, neurons, endothelial cells and immunocytes, including lymphocytes and macrophages. The current studies were performed to evaluate the role of SP in the pathogenesis of EMCV-induce myocarditis. SP levels were increased 61 fold in EMCV infected wild-type mice. EMCV infection resulted in 51% mortality at 14 days and a 1.56 fold increase in heart-to-body weight ratio that was accompanied by cardiac inflammation and necrosis and along with cardiomyocyte apoptosis and hypertrophy of surviving cells. In contrast, SP precursor knockout mice were completely protected from EMCV-mortality, cardiomegaly, cardiac inflammation and necrosis as well as cardiomyocyte apoptosis and hypertrophy. These results indicate that SP is essential for the pathogenesis of EMCV myocarditis and suggest that targeting this signaling pathway may be beneficial in viral myocarditis in humans.