Activation of nicotinic acetylcholine receptors (nAChRs) on inflammatory cells induces anti-inflammatory effects. The intracellular mechanisms that regulate this effect are still poorly understood. In neuronal cells, nAChRs are associated with phosphatidyl-inositol-3-kinase (PI3K). This enzyme, which can activate phospholipase C (PLC), is also present in monocytes. The aim of this study was to assess the role of these proteins in the signalling pathways involved in the anti-inflammatory effect of dimethyl-phenylpiperazinium (DMPP), a synthetic nAChR agonist, on monocytes and macrophages. The results indicate that PI3K is associated to alpha 3, 4 and 5 nAChR subunits in monocytes. PI3K inhibitors wortmannin and LY294002 abrogated the inhibitory effect of DMPP on lipopolysaccharide-induced TNF release by monocytes. Treatment with DMPP for 24 and 48 h provoked a mild PLC phosphorylation, which was blocked by the nAChR antagonist mecamylamine and reversed by PI3K inhibitors. Treatment of monocytes and alveolar macrophages with DMPP reduced the IP3 dependent intracellular calcium mobilization induced by platelet activating factor (PAF), an effect that was reversed by mecamylamine in alveolar macrophages. DMPP did not have any effect on PAF receptor expression. DMPP also inhibited the thapsigargin-provoked calcium release, indicating that the endoplasmic reticulum calcium stores might be depleted by the treatment with the nAChR agonist. Taken together, these results suggest that PI3K and PLC activation are involved in the anti-inflammatory effect of DMPP. PLC limited, but constant activation could induce the depletion of intracellular calcium stores, leading to the anti-inflammatory effect of DMPP.