Using immunohistochemical techniques, we studied IFN-gamma-producing cells (IFN-gamma-PC) in vivo during immune responses to thymus-independent type-2 (TI-2) Ag. Detection of IFN-gamma-PC in cryostat sections of spleen-tissue was performed with an enzyme labeled mAb directed against IFN-gamma. After TNP-Ficoll immunization, IFN-gamma-PC and TNP-specific antibody-forming cells (TNP-AFC) displayed similar kinetics reaching a maximum number at day 5 to 7. The IFN-gamma-PC were localized in the same compartment as TNP-AFC and a part of them in juxtaposition to TNP-AFC. Immunization with other TI-2 Ag resulted also in a significant increase of the number of IFN-gamma-PC. In a parallel experiment we found both in vivo and in an ELISA-spot assay a significant increase of the number of IFN-gamma-PC and IFN-gamma-spot-forming-cells, respectively, in spleens of mice 6 to 7 days after TNP-Ficoll immunization. Double staining of spleen sections for IFN-gamma and surface Ag revealed that 5 to 7 days after TNP-Ficoll immunization, +/- 40% of the IFN-gamma-PC expressed the MT4 Ag (CD4), +/- 50% the Lyt-2+ Ag (CD8) and +/- 10% the asialo-GM1 Ag (NK cell). This study represents the first description of the in vivo activity and characterization of IFN-gamma-PC during a TI-2 immune response. Moreover, the presented data confirm suggestions from in vitro investigations that IFN-gamma and T cells may play a direct role in the in vivo regulation of a primary immune response against a TI-2 Ag.