Tumor necrosis factor-α (TNF-α) binds to TNF-α receptors (TNFR) to produce a hexameric (TNF-α) 3 -(TNFR) 3 structure that stimulates apoptosis. We found by using ELISA that TNF-α binds to the glycosylphosphatidylinositol (GPI) anchor glycans of carcinoembryonic antigen, human placental alkaline phosphatase (hAP), and Tamm-Horsfall glycoprotein. These binding abilities were inhibited by 10 - 6 M mannose-6-phosphate. Treatment of hAP with mild acid and phosphatase, which releases the N-acetylglucosamine (GlcNAc) β1->phosphate->6 residue from the GPI-anchor glycan of hAP, abrogated the binding of TNF-α to hAP. Thus, TNF-α binds to the GlcNAcβ1->phosphate->6Man residue in GPI-anchor glycans. To investigate whether the carbohydrate-binding ability of TNF-α is related to its physiological functions, human lymphoma U937 cells were used. TNF-α stimulates U937 cell apoptosis in a dose-dependent manner and the presence of mannose-6-phosphate inhibited this. TNF-α-dependent tyrosine phosphorylation of several proteins in U937 cells was also diminished by mannose-6-phosphate. Phosphatidylinositol-specific phospholipase C-pretreatment also inhibited this tyrosine phosphorylation. These data suggest that TNF-α stimulates U937 cell apoptosis by forming a high-affinity nanomeric (TNF-α) 3 -(TNFR) 3 -(GPI-anchored glycan) 3 complex. The GPI-anchored glycoprotein involved remains to be identified.