This study was carried out to demonstrate sequential changes of antibody response to gp51 of BLV in bovine hosts injected with BLV-vaccine. BLV vaccine was prepared from culture fluids from FLK-BLV cells by treatment with 0.1% formalin for 48 hrs at 4 degrees C followed by ultrafiltration and lyophilization. Sterile vaccine containing 300 mg protein/ml, 1 mg of which was reactive by ELISA with monoclonal anti-BLV-glycopeptide up to 1:256 dilution, was injected intradermally with Freund's adjuvant into two 1-month-old Holstein calves seronegative for BLV. The first and second booster injections were given without adjuvant 3 and 15 weeks, respectively, after the initial injection. Sera collected weekly from these animals were analyzed to monitor development of antibody to BLV-gp51 by Western blotting and immunoferritin electron microscopy, as well as by ELISA to whole BLV and to BLV-gp51 partially purified by column chromatography. Antibody to BLV-gp51 was detected in sera collected 2 weeks after the initial injection, increased 2 weeks after the first booster, maintained its level during the following 10 weeks, and increased again 2 weeks after the second booster injection. Infectious BLV was not detected by syncytium-formation assay of lymphocytes collected 15 weeks after the initial injection. This study demonstrated sequential changes of anti-BLV-gp51 antibody elicited in bovine hosts subsequent to injection of formalin-treated BLV. Further analysis of bovine antibody to BLV-gp51 may help develop improved BLV-vaccine.