In order to analyze the molecular structures involved in T-cell recognition we developed an in vitro primary response against alloantisera bound to histocompatibility antigens in which nonimmune guinea pig T cells can be sensitized and subsequently challenged in tissue culture with antisera-treated macrophages. If macrophages were incubated with alloantisera directed against the I-region-associated (Ia) antigens of the guinea pig major histocompatibility complex (MHC) T cells could be sensitized to the antisera bound to macrophage Ia determinants. Anti-Ia-treated syngeneic macrophages in the first and second cultures elicited specific T-cell activation, as measured by increased DNA synthesis, to the antisera-induced immunogenic determinants. Similarly, antiIa-treated allogeneic macrophages also specifically stimulated T cells to antisera bound to allogeneic Ia determinants while reducing the mixed leukocyte reaction. Antisera to the B.1 antigens of the guinea pig MHC, the homologue of the mouse H-2K or H-2D antigens, also elicited specific T-cell activation that did not cross-react with that produced by the anti-Ia alloantisera. Furthermore, the anti-B.1-induced stimulation appeared to be associated with the Ia antigens of the macrophage used for priming since (2 x 13)F1 T cells sensitized with anti-B.1-treated parental macrophages could be restimulated only with the parental macrophage used for initial sensitization, and not with those of the other parent. Since the parental strain 2 and strain 13 guinea pigs express serologically identical B.1 antigens and differ only by Ia antigens of the MHC, this observation suggests that both B.1 and Ia antigens may be included in the immunogenic complex recognized by T cells. However, we cannot rule out the possibility that this restriction is due to other genetic differences between strain 2 and strain 13 guinea pigs that is unrelated to the I-region. We interpret these findings as showing that macrophage Ia antigens may serve to directly present antigens bound to the Ia molecule, and possibly indirectly aid in the presentation of antigens bound to other membrane components, such as the B.1 antigens.