Lymphoproliferative responses against a preparation of Eimeria bovis antigens (EBAg) were measured in E. bovis-immune and naive animals. Optimal lymphocyte responsiveness could be measured after 7 days of culture in the presence of antigen at a cell concentration of 2 X 10(5) cells per well. The specificity of the reaction was confirmed by limiting dilution analysis. Whereas immune peripheral blood mononuclear cells responded to EBAg (f = 1/18,824), naive cells did not (f = 0). The helper function of cells proliferating in response to EBAg was investigated by raising T-cell lines and a clonal population derived from a line. The T-cell line showed an enhanced reactivity to EBAg by limiting dilution analysis (f = 1/256) and was interleukin-2 dependent. Limiting dilution analyses indicated at least two populations of cells: one that was interleukin-2 restricted and antigen dependent and another that was antigen independent. Supernatants from T-cell lines and the clone were analyzed for the production of lymphokines after antigen stimulation. Minimal amounts of interleukin-2 were produced. The T-cell line produced both gamma interferon (IFN-gamma) (750 U) and IFN-alpha (1,250 U), whereas the clone produced IFN-gamma (1,250 U) only. Short-term (4-day) stimulation of immune cells by EBAg induced the production of IFN-gamma (600 U) and a non-IFN macrophage-activating lymphokine. We conclude that this macrophage-activating lymphokine is only produced after short-term culture and that further culture of T cells results in the proliferation of other clones producing other factors (such as IFN).