Difficulties in the isolation and long-term maintenance of bovine herpesvirus-1 (BHV-1) specific T-cell clones have hindered the analysis of bovine cell-mediated immune response to this virus. In an effort to identify the T-cell epitopes of the virus, bovine murine T-cell hybridomas specific for BHV-1 were generated as an alternative to T-cell clones. Peripheral blood lymphocytes from a calf immunized with BHV-1 were restimulated in vitro with the virus to generate bulk T-cell cultures. The antigen-specific T-cell-enriched bulk culture lymphocytes were fused with the T-cell receptor-deficient mutant of the murine thymoma cell line BW 5147. T-cell hybridomas were screened for their ability to produce interferon-gamma in response to BHV-1 stimulation. Hybridomas with various specificities were obtained. One of them was specific for the BHV-1 glycoprotein gI, two were specific for gIV, while three other hybridomas were specific for gIII. One hybridoma responded to stimulation with BHV-1, but not to any of the glycoproteins gI, gIII, or gIV, suggesting that proteins other than these major glycoproteins may be involved in the bovine T-cell response to BHV-1. Of these hybridomas, one was MHC Class I restricted, while all the others were Class II restricted.