Exposure of cells to liposomes results in the release of integral membrane proteins. However, it is still controversial whether the release is due to spontaneous protein transfer from cells to liposomes or shed vesicles released from cells. We investigated this issue in an erythrocyte-liposome system by examining the location of acetylcholinesterase (AChE, an integral membrane protein marker), cholesterol (erythrocyte membrane lipid marker), hemoglobin (cytosolic protein marker), and a nonexchangeable lipid marker in liposomes in a sucrose density gradient at high resolution. The density distribution showed that AChE is not transferred to the liposomes but is located on small (about 50 nm) light (10-20 wt % sucrose) or large (about 200 nm) heavy shed vesicles (more than 30 wt % sucrose). AChE in the light shed-vesicle fraction markedly increased even after its level in the heavy fraction reached a plateau. AChE was also released from isolated heavy shed vesicles and accumulated in the small light shed-vesicle fraction in the presence of liposomes. After incubation of spherical erythrocytes (morphological index, 5.0) with liposomes, AChE hardly appeared in the heavy shed-vesicle fraction, and the majority (>99%) appeared in the light shed-vesicle fraction, indicating that AChE is released from both the erythrocytes and heavy shed vesicles to the light shed-vesicle fraction, which becomes rich in AChE. Our results demonstrated for the first time that GPI-linked proteins do not spontaneously transfer from erythrocytes to liposomes. Our study also suggests that in vivo GPI-linked membrane proteins do not spontaneously transfer between cell membranes but that some catalyst is needed.