The susceptibility of medial and neointimal arterial smooth muscle cells (SMCs) to acute cytomegalovirus (CMV) infection was investigated in immunocompetent and immunocompromised rats. The left common carotid artery of all the rats was injured by balloon catheterization. On days 14 and 17 after injury, rats were either intravenously infected with a rat CMV (RCMV) or mock-infected. Active RCMV infection was shown in the neointima of the injured arteries of immunosuppressed rats, characterized by specific cytopathological changes in hematoxylin-eosin-stained sections and by the presence of early viral antigens and the presence of the virus at different stages of maturation at the electron-microscopic level. Viral genome was shown as well by use of in situ hybridization procedures. However, medial cells were hardly ever infected, and RCMV did not infect neointimal parts that were recovered by endothelial cells. No infection was seen in control right carotid arteries or in the injured arteries of immunocompetent rats. Acute intimal RCMV infection was accompanied by infiltration of inflammatory cells, predominantly mononuclear cells that stained positive with an ED-1 monoclonal antibody. The majority of infected neointimal cells were SMCs containing smooth muscle actin. No changes were found in medial or neointimal cross-sectional areas of the infected arteries. In the present study, an active RCMV infection of arterial SMCs was established in vivo, and it was concluded that neointimal SMCs are far more susceptible than are medial SMCs and that the absence of endothelium and immunosuppression are necessary conditions for arterial RCMV infection.