Toll-like receptors (TLRs) play an important role in innate immunity. They detect pathogen-associated receptor patterns (PAMPs) and initiate subsequent immune responses. Present studies investigate the influence of TLR2 ligands on leukotrienes (LT) formation in human monocytes. LTs are proinflammatory mediators derived from arachidonic acid (AA), which is released from membranes by phospholipase A(2) (PLA(2)) enzymes. Pretreatment of MM6 cells with the TLR2 ligands LTA, FSL-1, or Pam(3)CSK(4) resulted in an up to two- to threefold enhancement of ionophore-induced LT formation in a dose- and time-dependent manner and to an augmentation of ionophore-induced AA release with similar kinetics. Also in human peripheral blood mononuclear cells (hPBMC), TLR2 activators increased LT formation. Studies with PLA(2) inhibitors indicated that the increase of AA release is a result of enhanced activity of group IV cPLA(2) in MM6 cells. TLR2 ligands elicited the time-dependent activation of p38 MAPK and ERK1/2 pathways, which led to phosphorylation of cPLA(2)alpha at Ser(505). Simultaneous inhibition of p38 MAPK and ERK1/2 pathways prevented the increase of cPLA(2)alpha phosphorylation and the augmentation of AA release. TLR2 ligand-induced increase of AA release was blocked by a neutralizing anti-hTLR2 antibody, indicating that TLR2 mediates augmented cPLA(2) activation and subsequent LT biosynthesis.