Immunohistochemistry has been used to define the patterns and kinetics of IL-2, IL-4, and IFN-gamma production at the sites of Ag exposure and in the lymphoid tissues of immunized mice, and to examine the anatomic relationships between cytokine-producing T cells and various APC or Ag-stimulated B cells. The earliest detectable cytokine response to administration of a protein Ag in adjuvant was the appearance of IFN-gamma-producing NK cells at the site of immunization by day 3. T lymphocytes producing IL-2, IL-4, and IFN-gamma were initially detected in draining lymph nodes and spleen within 7 days after immunization, and IL-2-producing cells were present at the immunization site several weeks later. Thus, T cell activation is initiated within lymphoid tissues, and these cells migrate back to depots of Ag. The IFN-gamma produced by NK cells early after immunization may regulate the phenotype of the subsequent Ag-specific T cell response. Using a hapten to which the antibody response is oligoclonal and dominated by a single idiotype, Ag-stimulated (idiotype-producing) B cells could also be detected by immunohistochemistry. These B cells were present in the same areas of lymphoid tissues as cytokine-producing T lymphocytes. Two-color staining showed that idiotype-producing B cells were in close proximity to both IL-2- and IL-4-producing T cells, suggesting that T cells producing either of these cytokines could provide helper function for the B cells. Finally, after subcutaneous immunization with adjuvant, IL-2+ T cells were found adjacent to F4/80+ macrophages, suggesting that macrophages function as important APC in this response.