We deciphered the mechanisms of production of pro- and anti-inflammatory cytokines by adherent human blood mononuclear cells (PBMC) activated by lipopolysaccharide (LPS) or monophosphoryl lipid A (MPLA). Both LPS and MPLA induced tumor necrosis factor (TNF) production proved to be dependent on the production of interleukin-1β (IL-1β). Of note, MPLA induced IL-1β release in human adherent PBMCs whereas MPLA was previously reported to not induce this cytokine in murine cells. Both LPS and MPLA stimulatory effects were inhibited by Toll-like receptor-4 (TLR4) antagonists. Only monocytes activation by LPS was dependent on CD14. Other differences were noticed between LPS and MPLA. Among the different donors, a strong correlation existed in terms of the levels of TNF induced by different LPSs. In contrast, there was no correlation between the TNF productions induced by LPS and those induced by MPLA. However, there was a strong correlation when IL-6 production was analyzed. Blocking actin polymerization and internalization of the agonists inhibited MPLA induced TNF production while the effect on LPS induced TNF production depended on the donors (i.e. high TNF producers versus low TNF producers). Finally, conventional LPS, tolerized adherent PBMCs to TLR2 agonists, while MPLA primed cells to further challenge with TLR2 agonists.