Interleukin-12 (IL-12) and IL-4 are important immunoregulatory cytokines that determine the fate of naive T cells during antigen priming in mice and also influence cytokine synthesis by differentiated murine and human T cells. The roles of these cytokines in regulating the differentiation and effector function of bovine T cells are less well studied. We investigated the ability of human IL-12 and bovine IL-4 to modify cytokine expression by antigen-stimulated T cells from cattle immune to the protozoal parasites Babesia bovis and Babesia bigemina or reactive with Mycobacterium bovis purified protein derivative. Peripheral blood mononuclear cells (PBMC) were cultured with specific antigen and IL-4 or IL-12 for 1 week. Then viable lymphoblasts consisting of predominantly CD4+ T cells were restimulated with antigen and antigen-presenting cells (APC) with or without cytokine. Cell lines were cultured for several weeks, and following restimulation with antigen and APC in the absence of exogenous cytokine, the cell lines were analyzed for proliferation, interferon-gamma (IFN-gamma) production, and expression of IL-2, IL4-, IL-10, or IFN-gamma transcript levels using a quantitative competitive RT-PCR. IL-12 and IL-4 had no effect on the composition of CD4, CD8, or gammadelta T cells in the cell lines or on the level of antigen-induced proliferation. IL-12 stimulated enhanced levels of IFN-gamma protein and transcript expression in all cell lines, with no consistent effects on IL-2 or IL-4 expression. In two B. bovis-specific cell lines, IL-12 suppressed IL-10 expression. IL-4 had no consistent effect on expression of any cytokine. These results indicate the use of IL-12 as an adjuvant to enhance type 1 cytokine responses in cattle during antigen priming.