The synthetic immunomodulator muramyl dipeptide (MDP) has previously been shown to be able to synergize with lymphokines in enhancing the specific immune response of purified splenic B cells. Here, we have examined the effect of MDP in the process of B cell activation. We found that MDP alone was ineffective on the proliferation of either murine resting or in vivo activated B cells, whereas it enhanced the DNA synthesis of B cells once stimulated with anti-IgM antibodies. In agreement with these findings, cell cycle analysis by flow cytometry after acridine orange staining indicated that MDP by itself could not trigger the entry into the G1 phase of the cell cycle of nonactivated B lymphocytes. In contrast, in the presence of anti-IgM antibodies plus BSF-1, MDP promoted further cell cycle progression preferentially into the G1B compartment and also through the G2/M phase. A kinetic study showed that MDP was the most effective when added after the activation of B cells by anti-IgM antibodies plus BSF-1. Our present results suggest that functional receptors for MDP may be expressed on already cycling B cells.