Bacterial lipopeptides represent a group of bacterial compounds able to trigger the functions of cells of the innate immune response. Whereas diacylated lipopeptides are recognized by TLR2/6 dimers, triacylated lipopeptides were shown to act via TLR2/1 dimers. Although several previous studies dealt with the effect of the TLR2/1 ligand Pam(3)CysSK(4) on neutrophil granulocytes (PMN), it is still not clear whether TLR2/6 ligand lipopeptides can directly influence PMN functions. In the present study we used highly purified human neutrophils to investigate the direct effects of the diacylated mycoplasmal macrophage activating lipopeptide-2 (MALP-2) on the function of neutrophil granulocytes. After exposure to 10 ng/ml MALP-2 neutrophils acquired activated cell shape, secreted IL-8 and MIP-1beta and their phagocytic capacity was enhanced. Analysis of cell surface activation markers confirmed the activating effect of MALP-2, the expression of CD62L was downregulated whereas CD11b was upregulated on PMN after exposure to MALP-2. The constitutive apoptosis of PMN was inhibited after exposure to MALP-2. However, MALP-2 exerted only a short-term effect on the apoptosis of resting neutrophils, a longer lasting effect was observed after transendothelial migration. MALP-2 did not directly induce the production of reactive oxygen intermediates but primed PMN for a fMLP-induced oxidative burst. The migration of neutrophils was enhanced after treatment with MALP-2. This was due, however, to a chemokinetic rather than to a chemotactic effect. Pam(3)CysSK(4) also activated PMN, but in comparison to MALP-2, at higher concentrations. These findings suggest that diacylated lipopeptides are important microbial structures recognized by and acting on neutrophil granulocytes.