Cytochemical and immunological markers were used to phenotype the bovine lymphoblastoid cell lines BL-3*, EBL-1, and EBL-2. Southern blot experiments were also performed to test these lines for the presence of proviral bovine leukemia virus (BLV). The BL-3* cell line, originally derived from a case of sporadic bovine leukosis (non BLV-associated) but later infected with BLV in vitro, was found to contain BLV provirus and expressed the BLV-encoded envelope glycoprotein BLV-gp51. BL-3* cells express surface IgM and cytoplasmic IgM as well as class II antigens, and greater than 95% were negative for the T-cell markers B26A, sheep erythrocyte (E) receptors and alpha-naphthyl butyrate esterase (alpha-NB). BL-3* thus appears to be B-cell derived. EBL-1 and EBL-2 were derived from cows with enzootic bovine lymphosarcoma; however, these cell lines were found not to be infected with BLV. Phenotypically, EBL-1 and EBL-2 are mature T-cells, as they were positive for the B26A epitope, alpha-NB, and E receptors. These cell lines also express class II major histocompatibility antigens, indicating an activated state. The T-cell phenotype of EBL-1 and EBL-2 raises interesting questions concerning the possible role of other retroviruses and non BLV-infected transformed T-cells in the development of EBL tumors.