We quantified the CD4+ T cell proliferation specific for bovine herpesvirus 1 (BHV-1) in peripheral blood mononuclear cells from cattle. The stimulation index as detected in proliferative assays performed in the presence of BHV-1 antigen is highly variable in immune cattle. By using proliferative assays performed after negative selection we showed that, as expected, CD4+ T cells were the limiting cell type for antigen-induced proliferation. Neither B, gamma delta T nor CD8+ cells seemed to be involved. The limiting dilution method was established to obtain quantitative estimations, namely frequencies of specific T cells. When limiting dilution cultures were supplemented with interleukin-2 (IL-2), an IL-2 induced unspecific cell proliferation masked the specific T cell proliferation. Natural killer cells were not the major cell type involved, but CD4+ lymphocytes themselves seemed to respond to IL-2 irrespective of the presence of antigen. When cultures were performed without addition of IL-2, the frequency of BHV-1 specific proliferative T cells could be obtained by the difference between the frequency of proliferating cells calculated in the presence and absence of antigen. The method provides a sensitive and quantitative means to measure the T cell immune response to BHV-1 vaccine candidates.