Various infections sensitize to lethal shock by promoting hyperactivation of macrophages to LPS stimulation. Although macrophages are thought to be deactivated upon contact with apoptotic cells during Trypanosoma cruzi infection, T. cruzi infection also sensitizes mice to endotoxemia. Herein, we studied the mechanisms of sensitization to endotoxemia in T. cruzi-infected mice in order to solve the paradox. Live (but not fixed) trypomastigotes from various stocks sensitized mice to endotoxemia. Mice deficient in glycolipid recognition (TLR2(-/-) and CD1d(-/-)) were sensitized by infection to challenge with LPS. Infected mice hyperproduced TNF and IL-10 upon LPS challenge. Infected TNF-R1(-/-), macrophage migration inhibitory factor (MIF)(-/-) and IFN-gamma(-/-) mice were lethally sensitized, but infected TNF-R1(-/-) mice administered anti-MIF survived shock with LPS. Macrophages from infected mice hyperproduced TNF in response to LPS stimulation and displayed increased expression of TLR4 compared to non-infected controls. Treatment with the PGE(2) synthesis inhibitor acetylsalicylic acid (AAS) in vivo reduced parasitemia and enhanced LPS-stimulated production of TNF by macrophages, but the effect was less in infected mice than in normal mice. Nevertheless, AAS treatment did not increase the susceptibility of infected mice to sublethal shock with LPS. Our results point to independent MIF and TNF/TNF-R1 lethal pathways and suggest a role for hyperactivated macrophages in T. cruzi-sensitized LPS-induced shock.