There is some discussion as to whether glycosyl-phosphatidylinositol(GPI)-anchored proteins occur in microdomains in the cell membrane(.1,2) These putative microdomains have been implicated in processes such as sorting in polarized cells(3-5) and signal transduction(6-8), Complexes enriched in GPI-anchored proteins, cholesterol and glycosphingolipids have been isolated from cell membranes by using non-ionic detergents : these complexes were thought to represent a clustered arrangement of GPI-anchored proteins(9,10). However, results obtained when clustering of GPI-anchored proteins induced by antibodies or by detergents was prevented support the idea of a dispersed surface distribution of GPI-anchored proteins at steady state(11-13). Here we use chemical crosslinking to show that membrane microdomains of a GPI-anchored protein exist at the surface in living cells. This clustering is specific for the GPI-anchored form, as two transmembrane forms bearing the same ectodomain do not form oligomers. Depletion of membrane cholesterol causes the clustering of GPI-anchored proteins to break up, whereas treatment of cells with detergent substantially increases the size of the complexes. We find that in living cells these GPI-anchored proteins reside in microdomains consisting of at least 15 molecules, which are much smaller than those seen after detergent extraction.