Although the basis for the high mortality rate for patients with mixed bacterial infections is likely to be multifactorial, there is evidence for a synergistic effect of muramyldipeptide (MDP) with lipopolysaccharide (LPS) on the synthesis of proinflammatory cytokines by mononuclear phagocytes. In this study, co-incubation of human Mono Mac 6 cells with MDP and either LPS or peptidoglycan (PGN) resulted in an apparent synergistic effect on tumor necrosis factor-alpha (TNF-alpha) secretion. Although incubation of cells with MDP alone produced minimal TNF-alpha, it caused significant expression of TNF-alpha mRNA. These findings suggest that the majority of TNF-alpha mRNA induced by MDP alone is not translated into protein. Furthermore, simultaneous incubation of cells with MDP and either LPS or PGN resulted in TNF-alpha mRNA expression that approximated the sum of the amounts expressed in response to MDP, LPS, and PGN individually. These findings indicate that the apparent synergistic effect of MDP on TNF-alpha production induced by either LPS or PGN is due to removal of a block in translation of the mRNA expressed in response to MDP. In subsequent studies, the effects of MDP alone and its effect on the production of TNF-alpha by LPS and PGN were determined to be independent of CD14, Toll-like receptor 2, and Toll-like receptor 4. These findings indicate that MDP acts through receptor(s) other than those primarily responsible for transducing the effects of LPS and PGN. Successful treatment of patients having mixed bacterial infections is likely to require interventions that address the mechanisms involved in responses induced by a variety of bacterial cell wall components.