Bat lung cell culture NBL 12 Tb 1 Lu chronically infected with cell free bovine leukemia virus (BLV), designed by NBL BLV 2, produces virus proteins and showed the presence of cells with the BLV antigens as well as BLV-induced syncytia for more than 11 years. Virus replication was abundant and increased with passage in NBL BLV 2 cell line and was moderate but decreased with passage in fetal lamb splenic cell line chronically infected with BLV (FLS NI 1228). The amount of virus proteins released in culture fluid by NBL BLV 2 cultures growing on roller bottle was 32.6-37.5 micrograms/ml, 42.7 micrograms/ml at passage 16-19 and 24-28, respectively. Studies in our laboratory have demonstrated that the virus protein production of NBL BLV cultures was 12.5 micrograms/ml in Falcon flasks (static), 26.3-52.8 micrograms/ml in roller bottle (rolling), 60.2 micrograms/ml in roller bottle with microcarriers and 9.82 microcarriers suspension (fermentor) as monolayer cell culture systems. On the monolayer cultures tested NBL BLV 2 cell line showed a progressive increase of virus production that reached high levels, for over 8 years and continues to release large amounts of virus. NBL BLV 2 cell line is also free of adventitious agents, it is easy to maintain and contains only small amount of cell debris, it provides a suitable source of cell free and cell associated bovine leukemia virus for research and production purposes.