We have previously developed a cell-free assay from rat skeletal muscle which displayed in vitro glucose transporter 4 (GLUT-4) transfer from large to small membrane structures by the addition of a cytosolic protein fraction. By combining protein fractionation and the in vitro GLUT-4 transfer assay, we have purified a glycosylphosphatidylinositol phospholipase D (GPI-PLD) which induces transfer of GLUT-4 from small to large membranes. The in vitro GLUT-4 transfer was activated and inhibited by suramin and 1,10 phenanthroline (an activator and an inhibitor of GPI PLD activity, respectively). Furthermore, upon purification of the GLUT-4 transporter protein, the protein displayed an elution profile where the molecular weight was related to the charge, suggesting the presence or absence of phosphate. Secondly, by photoaffinity labeling of the purified GLUT-4 with [¹²⁵I] 3-(trifluromethyl)-3-(m-[¹²⁵I]iodophenyl) diazirine both labeled phosphatidylethanolamine and fatty acids (constituents of a GPI link) were recovered. Third, by using phase transition of triton X-V114, the purified GLUT-4 was found to be partly detergent resistent, which is a known characterstic of GPI-linked proteins. Fourth, the purified GLUT-4 protein was recognized by an antibody raised specificly against GPI links. In conclusion, GLUT-4 containing vesicles may be released from a membrane compartment by action of a GPI PLD.