The TNF-α-inducing capacity of different trypanosome components was analyzed in vitro, using as indicator cells a macrophage cell line (2C11/12) or peritoneal exudate cells from LPS-resistant C 3 H/HeJ mice and LPS-sensitive C 3 H/HeN mice. The variant-specific surface glycoprotein (VSG) was identified as the major TNF-α-inducing component present in trypanosome-soluble extracts. Both soluble (sVSG) and membrane-bound VSG (mfVSG) were shown to manifest similar TNF-α-inducing capacities, indicating that the dimyristoylglycerol (DMG) compound of the mfVSG anchor was not required for TNF-α triggering. Detailed analysis indicated that the glycosyl-inositol-phosphate (GIP) moiety was responsible for the TNF-α-inducing activity of VSG and that the presence of the GIP-associated galactose side chain was essential for optimal TNF-α production. Furthermore, the results showed that the responsiveness of macrophages toward the TNF-α-inducing activity of VSG was strictly dependent on the activation state of the macrophages, since resident macrophages required IFN-γ preactivation to become responsive. Comparative analysis of the ability of both forms of VSG to activate macrophages revealed that mfVSG but not sVSG stimulates macrophages toward IL-1α secretion and acquisition of LPS responsiveness. The priming activity of mfVSG toward LPS responsiveness was also demonstrated in vivo and may be relevant during trypanosome infections, since Trypanosoma brucei-infected mice became gradually LPS-hypersensitive during the course of infection. Collectively, the VSG of trypanosomes encompasses two distinct macrophage-activating components: while the GIP moiety of sVSG mediates TNF-α induction, the DMG compound of the mfVSG anchor contributes to IL-1α induction and LPS sensitization.