Glycosylphosphatidylinositol (GPI)-anchored cell surface proteins are widely expressed in tissues, including cells of immunohematopoietic origin. Cross-linking of GPI-linked proteins on T lymphocytes, such as Thy-1 (CD90), Ly-6 A/E, CD48, CD59 and others, induces T-cell mitogenesis. Similar to cross-linking with T-cell receptor (TcR)-specific antibodies, ligation of GPI-anchored proteins induces an intracellular flux of calcium, an up-regulation of activation-associated cell surface proteins and the elaboration of growth-promoting lymphokines. These events are dependent on p56(lck)-mediated tyrosine phosphorylation of substrates. GPI-linked proteins are constitutively clustered in sphingolipid-rich membrane domains. Actin-driven rearrangements of the cytoskeleton are probably responsible for the physical approximation of TcR and GPI-anchored proteins in mature immunological synapses. Functionally, GPI-linked proteins can supplant for signal I and productively collaborate with CD28 to fully activate T cells.