Bovine leukemia virus (BLV) infection of cattle is a common but inapparent retrovirus infection from which less than 5% of infected cattle manifest clinically with lymphoma. During the course of life-long infection, cattle maintain a high anti-BLV titer during an apparent latent infection of B cells from which proviral DNA but not transcripts can be detected. To investigate if BLV infection is truly latent and restricted only to B lymphocytes, peripheral blood leukocytes were purified from cattle with naturally acquired BLV infection with various stages of subclinical and clinical disease. These cells were purified into populations of polymorphonuclear cells, monocytes and subsequently B cells and T cells by fluorescent-activated cell sorting. Southern blot analysis revealed the presence of provirus in monocytes and B cells but not T cells. Secondly, the ability of provirus containing cell types to express BLV antigens was then confirmed by mitogen stimulation in vitro. Subsequently, the pattern of expression of BLV antigens was studied in situ in tissue sections to determine the location of BLV antigen expressing cells in vivo. Antigen expressing cells were infrequent and solitary in intrafollicular and marginal zone cells of architecturally normal lymph nodes of chronic, BLV-infected cattle. These results demonstrate that BLV persists in cells of the monocyte/macrophage lineage in addition to B lymphocytes and that expression of BLV antigens in cattle doses occur but rarely in lymph nodes of BLV infected cattle. The high frequency of provirus containing cells and infrequent expression of BLV antigen in vivo suggests that BLV expression is not truly latent but highly regulated, possibly triggered by rare events in host lymphoid tissue.