We have used MLR to assess the cell interactions that enable human PBL stimulated with irradiated, allogeneic PBL to give rise to allospecific, IFN-gamma-producing CD4 T cells. We were able to generate alloantigen-specific, IFN-gamma-producing T cells from peripheral blood. The responder and stimulator cell numbers required for the optimal generation of such cells, enumerated using an enzyme-linked immunospot assay, were separately determined for various combinations of responders and stimulators. The number of antigen-specific, IFN-gamma-producing cells generated per 10(6) responder cells, seeded at the initiation of culture, is critically dependent on the density of the responder cells, with minimal generation of IFN-gamma-producing T cells at low responder densities. These and further observations with fractionated responding PBL show that CD4 T-cell/CD4 T-cell interactions, which are completely independent of CD8() T cells, are necessary for the in vitro generation of alloantigen-specific, IFN-gamma-producing CD4 T cells.