A method is described for stimulating proliferation in primed populations of murine T lymphocytes using antigen bound to mitomycin-C-treated spleen cells. This form of antigen presentation appears to be an active process because heat-killed spleen cells are ineffective, and because genetic similarity at the major histocompatibility complex (MHC) between the responder T cells and the presenting spleen cells is required for effective interactions. At all times examined, from day 3 to day 6 of the proliferative response, syngeneic spleen cells presented antigen better to peritoneal exudate T-lymphocyte-enriched cells (PETLES) than semisyngeneic F(1) spleen cells, which in turn could present antigen better than totally allogeneic spleen cells. Spleen cell mixing experiments demonstrated that these genetic restrictions were not the result of suppression by the ongoing mixed lymphocyte reactions (MLR) in the allogeneic and F(1) cases. Furthermore, incompatibility at the Mls locus generated a strong MLR but failed to prevent antigen presentation if the spleen cells and PETLES were compatible. Genetic mapping studies demonstrated that compatibility at only the I-A subregion of the MHC was sufficient for effective presentation of the antigen, dinitrophenylated ovalbumin. Compatibility at only the K region, or the K and D regions was not sufficient. These results support the concept that functional activation of primed, proliferating T lymphocytes requires the participation of gene products coded for by the I region of the MHC. This conclusion is consistent with a growing body of evidence which suggests that most T cells recognize antigen in association with MHC gene products.