MHC unrestricted, antigen nonspecific killing by CD4^+ T-cells against virally-infected target cells was induced following cross-linking of CD4 molecules. The cytotoxicity of antibody-activated CD4^+ T-cells was abolished by genistein (4`,5,7-trihydroxyisoflavone), a protein tyrosine kinase (PTK) inhibitor, but not by H-7, a protein kinase C (PKC) inhibitor. Genistein-treated human or bovine peripheral blood CD4^+ T-cells lacked PTK activity and failed to kill virally-infected target cells even after cross-linking of CD4 molecules. The cross-linking of CD4 molecules did not induce effector cell proliferation or the transcription of TNF β. TNF β synthesis was up-regulated by incubating antibody activated effector cells with bovine herpesvirus type 1 (BHV-1) infected D17 target cells. Anti-TNF β antibody partially abrogated direct effector cell-mediated antiviral cytotoxicity. On the other hand, this antibody effectively neutralized antiviral activity of effector and target cell culture supernatants against BHV-1 infected D17 cells. The inhibition level of the antiviral activity by the antibody was dependent on effector and target cell ratio. These findings have importance to define the mechanisms of how CD4 cytotoxic cells control viral infection