The association of glycosylphosphatidylinositol (GPI)-anchored cell surface glycoproteins with Src-family protein tyrosine kinases was analysed in intact T lymphocyte plasma membranes. Following subcellular fractionation without detergent, 25% of the recovered plasma membranes were light density vesicles enriched in GPI-anchored glycoproteins and sphingolipids (GPI domains), while the remainder behaved as heavier density vesicles containing equal amounts of lipids and proteins. Qualitatively similar lipids were found in both vesicle types, but only light density vesicles made of 65-75% lipids yielded a Triton X-100 resistant, sedimentable fraction containing GPI-linked glycoproteins and sphingolipids. The GPI-rich vesicles phosphotyrosylated an exogenous substrate as efficiently as the denser vesicles, despite a low Lck and Fyn kinase content. Likewise, these kinases were more efficiently phosphorylated in GPI domains than in denser vesicles. GPI domains thus could constitute plasma membrane "hot spots" where associated Src kinases assume an optimally active conformation that contributes to signaling via GPI-anchored cell surface glycoproteins.