Membrane-associated decay accelerating factor (DAF) of human erythrocytes (Ehu) was analyzed for a C-terminal glycolipid anchoring structure. Automated amino acid analysis of DAF following reductive radiomethylation revealed ethanolamine and glucosamine residues in proportions identical with those present in the Ehu acetylcholinesterase (AChE) anchor. Cleavage of radiomethylated 70-kilodalton (kDa) DAF with papain released the labeled ethanolamine and glucosamine and generated 61- and 55-kDa DAF products that retained all labeled Lys and labeled N-terminal Asp. Incubation of intact Ehu with phosphatidylinositol-specific phospholipase C (PI-PLC), which cleaves the anchors in trypanosome membrane form variant surface glycoproteins (mfVSGs) and murine thymocyte Thy-1 antigen, released 15% of the cell-associated DAF antigen. The released 67-kDa PI-PLC DAF derivative retained its ability to decay the classical C3 convertase C4b2a but was unable to membrane-incorporate and displayed physicochemical properties similar to urine DAF, a hydrophilic DAF form that can be isolated from urine. Nitrous acid deamination cleavage of Ehu DAF at glucosamine following labeling with the lipophilic photoreagent 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine ([125I]TID) released the [125I]TID label in a parallel fashion as from [125I]TID-labeled AChE. Biosynthetic labeling of HeLa cells with [3H]ethanolamine resulted in rapid 3H incorporation into both 48-kDa pro-DAF and 72-kDa mature epithelial cell DAF. Our findings indicate that DAF and AChE are anchored in Ehu by the same or a similar glycolipid structure and that, like VSGs, this structure is incorporated into DAF early in DAF biosynthesis prior to processing of pro-DAF in the Golgi.