Incubation of adherent cells derived from peripheral blood mononuclear cells of cattle naturally infected with bovine leukemia virus (BLV) led to the establishment of three, persistently infected, primary cell cultures. These cultures were obtained exclusively from animals exhibiting persistent lymphocytosis, and not from uninfected or infected, hematologically normal cattle. The cells contained monoclonally integrated, full length BLV provirus, indicating that each culture resulted from clonal expansion of a single cell. They expressed high levels of all BLV specific mRNAs and showed intracellular reactivity to antibodies directed to viral gag and env proteins. Viral particle morphogenesis was highly restricted as determined by low levels of reverse transcriptase activity in cell supernatants and the paucity of viral particles on the cell surface. Analysis of cellular antigenic determinants, using monoclonal antibodies to bovine leukocyte differentiation and major histocompatibility complex antigens, was inconclusive. Cytochemical, morphologic, and ultrastructural analyses were consistent with endothelial cells and they exhibited the distinctive functional capacity of endothelial cells derived from specialized postcapillary venules, which constitute sites of lymphocyte extravasation. These data suggest that infection of these endothelial cells may be involved in the development of persistent lymphocytosis in BLV-infected animals.