SummaryWe established 9 cell lines from 63 tumor cases of enzootic bovine leukosis and studied their properties. Cells of all lines formed small clumps and floated in culture medium, indicating growth. Four of the 9 cell lines were surface immunoglobulin (SIg)‐positive, but the remaining 5 line cells were negative for SIg or, if SIg was detected, the percentage of SIg‐positive cells was very low. Tests for the properties of the cells with monoclonal antibodies to lymphocytes revealed that the established line cells are B‐lymphocytes. Morphological observation also revealed that they had the morphology of B‐lymphoblastic cell. The results of E and EAC rosette assay were negative, but 6 of 8 cell lines were positive for EA rosetting. All the 9 cell lines reacted with MoAb C‐143, which recognizes the tumor‐associated antigen (TAA) of the EBL tumor cell. All 9 cell lines produced bovine leukosis virus (BLV). These results suggest that the 9 cell lines are tumor cells derived from B‐lymph