Toxoplasma gondii is a ubiquitous parasitic protozoan, which causes congenital infectious diseases as well as severe encephalitis, a major cause of death among immune-deficient persons, such as AIDS patients. T. gondii is normally controlled by the immune system of healthy individuals, leading to an asymptomatic infection. T. gondii triggers early cytokine production, which, to a certain extent, protects the host against replication of tachyzoites, the infective form of the parasite. Glycosylphosphatidylinositols (GPIs) constitute a class of glycolipids that have various functions, the most fundamental being to link proteins to eucaryotic cell membranes. GPIs are involved in the pathogenicity of other protozoan parasites and are known to induce tumor necrosis factor-alpha (TNF alpha) production. We show that GPIs highly purified from T. gondii tachyzoites, as well as their core glycans, induce TNF alpha production in macrophages. A chemically synthesized GPI of T. gondii lacking its lipid moiety, GPIa, has the same effect as the natural GPIs, whereas a chemically synthesized molecule with dialkylglycerol instead of diacylglycerol as lipid moiety, GPIb, does not induce TNF alpha production. Moreover, GPIb inhibits the TNF alpha production induced by T. gondii GPI or by GPIa. The core glycan prepared from the two chemically synthesized molecules activates macrophages, showing that the lipid moiety may regulate signaling. Stimulation of macrophages with GPIs of T. gondii results in activation of the transcription factor NF-kappa B, which is inhibited by the chemically synthesized GPIb, suggesting the involvement of NF-kappa B in TNF alpha gene expression. Our results support the idea that T. gondii GPIs are bioactive factors that participate in the production of TNF alpha during toxoplasmal pathogenesis.