Mouse spleen cells cultured with muramyl dipeptide (MDP) release factors (SMF, or splenic macrophage factors) which enhance the in vitro plaque-forming cell (PFC) response to the T-dependent antigen SRBC (sheep red blood cells). The present study substantiates previous results and shows that T cells were the target cells of such conditioned medium. We investigated which T-cell subpopulations had their activities modified by these factors. In the presence of SMF, T cells educated in vivo elicited enhanced PFC responses to both specific educating antigen and an unrelated antigen (during a bystander response). This could indicate that SMF potentiates Th2-cell activity. Moreover, in vivo matured but unprimed thymocytes, which are unable to cooperate with virgin B cells in an in vitro T-dependent response, are able to do so when SMF is added in cooperative cultures. It is concluded that MDP-conditioned medium allows the maturation/differentiation of a subset of T cells to the point where they can be stimulated by the antigen.