Soluble mediators such as interleukin-1, tumor necrosis factor alpha (TNF-), and inducible nitric oxide synthase (iNOS) produced from activated macrophages play an important role in the destruction of pancreatic  cells in mice infected with a low dose of the D variant of encephalomyocarditis (EMC-D) virus. The tyrosine kinase signaling pathway was shown to be involved in EMC-D virus-induced activation of macrophages. This investigation was initiated to determine whether the Src family of kinases plays a role in the activation of macrophages, subsequently resulting in the destruction of  cells, in mice infected with a low dose of EMC-D virus. We examined the activation of p59/p56^Hck, p55^Fgr, and p56/p53^Lyn in macrophages from DBA/2 mice infected with the virus. We found that p59/p56^Hck showed a marked increase in both autophosphorylation and kinase activity at 48 h after infection, whereas p55^Fgr and p56/p53^Lyn did not. The p59/p56^Hck activity was closely correlated with the tyrosine phosphorylation level of Vav. Treatment of EMC-D virus-infected mice with the Src kinase inhibitor, PP2, resulted in the inhibition of p59/p56^Hck activity and almost complete inhibition of the production of TNF- and iNOS in macrophages and the subsequent prevention of diabetes in mice. On the basis of these observations, we conclude that the Src kinase, p59/p56^Hck, plays an important role in the activation of macrophages and the subsequent production of TNF- and nitric oxide, leading to the destruction of pancreatic  cells, which results in the development of diabetes in mice infected with a low dose of EMC-D virus.