Normal cellular prion protein (PrP(C)) and decay-accelerating factor (DAF) are glycoproteins linked to the cell surface by glycosylphosphatidylinositol (GPI) anchors. Both PrP(C) and DAF reside in detergent insoluble complex that can be isolated from human peripheral blood mononuclear cells. However, these two GPI-anchored proteins possess different cell biological properties. The GPI anchor of DAF is markedly more sensitive to cleavage by phosphatidylinositol-specific phospholipase C (PI-PLC) than that of PrP(C). Conversely, PrP(C) has a shorter cell surface half-life than DAF, possibly due to the fact that PrP(C) but not DAF is shed from the cell surface. This is the first demonstration that on the surface of the same cell type two GPI-anchored proteins differ in their cell biological properties.