Synthetic muramyldipeptide (MDP), the minimum structural moiety of bacterial peptidoglycan for adjuvant and related activities, sensitized mice for two types of lethal shock induced by lipopolysaccharide (LPS): an early anaphylactoid shock and late endotoxin shock. In relation to the late reaction in MDP-primed mice, enhanced production of inflammatory cytokines was induced in response to various bacterial components. MDP showed a priming effect in mice not only when administered parentally but also via the oral route. MDP activated human monocytic THP-1 cells in a CD14-, Toll-like receptor 2 (TLR2)- and TLR4-independent manner to increase expression of MyD88, a common adaptor and signaling molecule for TLRs, and exhibited synergistic cytokine-inducing effects with TLR4 agonists (LPS, synthetic lipid A), TLR2 agonist (synthetic lipopeptide), and TLR9 agonist (bacterial CpG DNA) in THP-1 cells in culture. Consistent with these findings, MDP primed TLR2 knockout mice as well as wild-type controls, but not TLR4-mutated C3H/HeJ mice, to enhance production of tumor necrosis factor-α upon stimulation with synthetic lipid A. In contrast to the BCG- and Propionibacterium acnes-priming system, MDP primed mice in an interferon-γ-independent manner. Further studies are required to elucidate the mechanisms of the synthetic and priming activities of MDP for various bacterial components.