The C57Bl/6-derived T cell line, L12-R4, produced murine interferon-gamma (IFN gamma) in response to mitogenic stimulation by phorbol myristate acetate (PMA) or concanavalin A (Con A), but not by staphylococcal enterotoxin A (SEA). Low levels of IFN gamma were produced by SEA stimulation of L12-R4 cells cocultured with C57Bl/6 bone marrow macrophages (BMM). Significantly increased yields of IFN gamma resulted from 48-hour pretreatment of the BMM with recombinant IFN gamma (100 U/ml) prior to coculture. Polyclonal anti-IFN gamma and anti-IFN alpha/beta were used to characterize the interferon as IFN gamma. Paraformaldehyde (0.1%) treatment of IFN gamma-pretreated BMM did not affect IFN gamma production, suggesting that processing of SEA was not required. IFN gamma treatment of BMM resulted in significantly increased expression of immune-associated (Ia) antigen as determined by flow cytometric analysis, suggesting that the accessory cell role of BMM involved Ia antigen. Polyclonal anti-Ia antibody selectively inhibited the production of IFN gamma by SEA-stimulated whole spleen cell cultures, consistent with the necessity of Ia antigen for BMM help in SEA induction of IFN gamma. More interestingly, induction of IFN gamma. These findings suggest that Ia antigen is necessary for BMM accessory function in SEA induction of IFN gamma. More interestingly, the results implicate class II molecules in a positive feedback loop for IFN gamma production by SEA.