We have established four cell lines in vitro from peripheral leukemic cells of four independent enzootic bovine leukosis (EBL) cattle. All cell lines exhibited differentiated B-cell characters. Two of them produced infectious bovine leukemia virus (BLV), but the others did not. Nonproducer cell lines contained single copies of defective BLV proviral genomes with the same integration profiles as the uncultured cells. On the other hand, numerous proviral copies were detected in producer cell lines. One of the producer cell lines, BL407, whose original uncultured cell contained complete and defective proviral genomes retained the original two copies and had increased only complete genomes in different integration sites after long term culture. These findings suggest that the monoclonal leukemic cells from EBL cases are preferentially established in vitro irrespective of their proviral structures, and the producer B-lymphoid cells amplify their proviral copies by reinfection with viruses re-expressed from the cells during in vitro cultivation.