Several proteins are attached to the cell membrane by a glycosyl- phosphatidylinositol (GPI) anchor. In this report, we show that during vesiculation of human RBCs in vitro, two of these proteins, acetylcholinesterase and decay accelerating factor, redistribute on the cell surface and become enriched in the released vesicles. As a result, the remnant cells are depleted of these proteins. We suggest that alterations in the architecture of the RBC membrane that precede vesiculation lead to selective polarization of GPI-anchored proteins within the domain of the membrane destined to become a vesicle. Since vesiculation occurs in many cell types, and if the loss of GPI-anchored proteins accompanies this process, it may have important biologic significance.