Short-term lymphocyte cultures from bovine leukemia virus (BLV)-infected cattle were tested for BLV-associated antigens at various times after incubation. Several immunologic methods were used, including fluorescent antibody tests, immunodiffusion, and radial immunodiffusion. Antigens were not detected in uncultured lymphocytes. The BLV-associated antigens were detected as early as 3 hours, with maximum antigen production occurring at 18 to 24 hours after incubation. These results indicate that culturing of lymphocytes in vitro is necessary for the expression of the virus.