Previous studies of cytomegalovirus (CMV) retinitis have failed to definitively explain the exact mechanism by which CMV gains access to and initiates infection in the retina. Proposed theories have included leakage of the virus through vessels with altered permeability, with subsequent infection of surrounding glial cells. In an attempt to shed further light on this subject, a histopathologic examination of 30 autopsy eyes from patients with known systemic CMV disease was carried out using light microscopy, immunohistochemical and immunofluorescent techniques, and in-situ hybridization. Dual-staining methods were used to identify the exact cell type showing the presence of CMV antigens, namely vascular endothelial cells, glial cells, neuronal cells, and/or leukocytes. In those eyes with CMV retinitis, the sites of full-thickness retinal necrosis revealed viral presence mostly within Muller cells and perivascular glial cells, with focal areas of positive staining within retinal pigment epithelial cells (RPE) and neuronal cells. The retinal capillaries were devoid of endothelial cells in these areas. Adjacent to regions of full-thickness necrosis, some vessels showed the presence of a viral antigen within the endothelial cells. These findings suggest that retinal vascular endothelial cells can be infected with CMV. It can further be hypothesized that infection of vascular endothelial cells leads to infection of the surrounding glial and neuronal cells, with eventual spread to the RPE. Endothelial cells might not be present in areas of full-thickness necrosis due to mechanical forces from adjacent blood flow resulting in the sloughing of these cells.