Although acetylcholine (ACh) is well known for its neurotransmitter function, recent studies have indicated that it also functions as an immune cytokine that prevents macrophage activation through a 'cholinergic (nicotinic) anti-inflammatory pathway'. In this study, we used the macrophage-like U937 cells to elucidate the mechanisms of the physiologic control of cytokine production by auto/paracrine ACh through the nicotinic class of ACh receptors (nAChRs) expressed in these cells. Stimulation of cells with lipopolysaccharide up-regulated expression of alpha1, alpha4, alpha5, alpha7, alpha10, beta1 and beta3 subunits, down-regulated alpha6 and beta2 subunits, and did not alter the relative quantity of alpha9 and beta4 mRNAs. Distinct nAChR subtypes showed differential regulation of the production of pro- and anti-inflammatory cytokines. While inhibition of the expression of the TNF-alpha gene was mediated predominantly by the alpha-bungarotoxin sensitive nAChRs, that of the IL-6 and IL-18 genes-by the mecamylamine-sensitive nAChRs. Both the Mec- and alphaBtx-sensitive nAChRs regulated expression of the IL-1beta gene equally efficiently. Upregulation of IL-10 production by auto/paracrine ACh was mediated predominantly through alpha7 nAChR. These findings offer a new insight on how nicotinic agonists control inflammation, thus laying a groundwork for the development of novel immunomodulatory therapies based on the nAChR subtype selectivity of nicotinic agonists.