Glycosylphosphatidylinositol (GPI)-anchored proteins participate in many cell surface functions; however, the molecular associations of these lipid-linked proteins within the plasma membrane are not well understood. Recent biochemical analyses of detergent insoluble membrane fractions have suggested that GPI-anchored proteins may be associated with glycosphingolipid (GSL)-enriched domains that also contain cholesterol and signaling molecules such as Src family kinases and, in some cases, caveolae. The movements of two components of the putative GSL-enriched domains, Thy-1, a GPI-anchored protein, and GM1, a GSL, were followed with single particle tracking on C3H 10T1/2 cell surfaces and categorized into four modes of lateral transport, fast diffusion, slow anomalous diffusion, diffusion confined to 325-370 nm diameter regions, and a fraction of molecules that was essentially stationary on the 6.6 s time scale. Longer observations (60 s) showed that Thy-1 and GM1 are transiently confined for 7-9 s to regions averaging 260-330 nm in diameter. Approximately 35-37% of both Thy-1 and GM1 undergo confined diffusion, whereas only 16% of fluorescein phosphatidylethanolamine, a phospholipid analog which is not expected to be found in the GSL domains, experience confined diffusion to regions averaging approximately 230 nm in diameter. Further, when glycosphingolipid expression was reduced approximately 40% with the glucosylceramide synthase inhibitor, d-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol, the percentage of trajectories exhibiting confinement and the size of the confining domain for Thy-1 were reduced approximately 1.5-fold. In contrast, extraction of cells with Triton X-100 leaves the fraction of molecules confined and the domain sizes of Thy-1 and GM1 unchanged. Our results are consistent with the preferential association of GPI-anchored proteins with glycosphingolipid-enriched domains and suggest that the confining domains may be the in vivo equivalent of the detergent insoluble membrane fractions.