Human cytomegalovirus (HCMV) infection is associated with a variety of systemic and neurologic diseases. In vitro HCMV growth is usually studied in fibroblasts, while in vivo HCMV growth is frequently observed in a wide variety of cell types including glia, neurons, and human brain capillary endothelial (HBCE) cells. To examine the biology of HCMV in HBCE cells, we have established a procedure for isolating these cells from human brain temporal lobectomies. Greater than 99.0% of these cultured cells were identified as HBCE cells on the basis of positive staining for factor VIII-related antigen-Von Willebrand's factor (F VIII) and Ulex Europaeus agglutinin I (UEA I). HCMV antigens were detected by immunocytochemistry in HBCE cells after infection with strain AD 169. Intracellular virions were observed in infected cells by electron microscopy and infectious virus was released from HBCE cells. In addition, infected cells were confirmed as endothelial cells by double staining with antibodies to F VIII and HCMV.