In T cells, glycolipids, glycoproteins attached to the membrane via a glycosylphosphatidylinositol (GPI) anchor, and Src-like tyrosine kinases are highly enriched in a membrane fraction resistant to solubilization by nonionic detergents. We have investigated the distribution of CD4 in T-cell membranes and found that approximately 10% of the CD4 co-receptor is associated with detergent-insoluble membrane microdomains, whilst the remaining 90% is in soluble membranes. Moreover, approximately 60% of the "insoluble CD4" is present in membrane microdomains containing GPI-anchored proteins and high glycolipid-dependent kinase activity, whereas the remaining 40% displays no association with GPI-anchored proteins and lacks glycolipid-associated kinase activity These results indicate that CD4 segregates at least into three different membrane microenvironments: 1) soluble membranes; 2) insoluble membrane microdomains containing GPI-anchored proteins; and 3) insoluble membrane microdomains devoid of GPI-anchored proteins. The level of CD4 in insoluble membranes was not modified upon triggering activation by T-cell receptor-crosslinking but detectable amounts of CD3 subunits were recruited into these specialized membranes under those conditions. The physical separation of CD4 into different membrane microenvironments raises the possibility of that some of the multiple functions of CD4 might segregate into distinct types of lipid microenvironment. The fact that components of T-cell receptor/CD3 complex were recruited into insoluble membranes upon stimulation is consistent with the CD4 present in this membrane fraction might participate in T-cell receptor-triggered activation events.