Cultured CD4+ cells are responsible for transfer of adoptive murine experimental hypersensitivity pneumonitis (EHP) (ARRD 1992; 146:1582-8). To characterize interactions that occur in vitro that result in cells able to transfer EHP, we added either antibody to IFN-gamma, antibody to IL-2, or 30 or 300 micrograms/ml IFN-gamma at the onset of 72-hour culture of C3H/HeJ spleen cells from either M. faeni or ovalbumin (control) sensitized donors with 30 micrograms/ml Micropolyspora faeni. We determined the phenotype of cells after culture and the amount of IL-2 or IFN-gamma in the culture supernatants, transferred cells to naive recipients, challenged the recipients intratracheally with M. faeni, and determined the extent of pulmonary inflammatory changes 4 days thereafter. Substantial amounts of IL-2 and IFN-gamma were detected in supernatants of cultures from M. faeni-sensitized animals, and lesser amounts were detected in culture supernatants from ovalbumin-sensitized donors. Treatment of cultures of M. faeni-sensitized cells with antibody to IL-2 or IFN-gamma blocked or reduced measurable IL-2 or IFN-gamma for the duration of culture. Treatment with IFN-gamma blocked increased levels of IL-2 at 48 and 72 hours of culture. Cultured M. faeni-sensitized cells adoptively transfer EHP. Cells from cultures depleted of either IL-2 or IFN-gamma or supplemented with IFN-gamma could transfer EHP equally well. We conclude that in vitro maturation of cells capable of adoptive EHP is not dependent on soluble IL-2 or IFN-gamma and is not altered by exogenous IFN-gamma.