The role of Rab4, a small molecular weight GTP binding protein implicated in endosomal/plasma membrane (PM) recycling, in the translocation of the GLUT4 transporter in rat skeletal muscle was studied. Muscle membranes, prepared by subcellular fractionation of control and insulin treated rat skeletal muscle, were subjected to SDS/PAGE and immunoblot analyses. Insulin treatment caused an increase in GLUT4 in a plasma membrane (PM) enriched fraction from an intracellular membrane (IM) fraction. Immunoprecipitation of GLUT4 vesicles from the IM compartment revealed that Rab4 could be coprecipitated. Importantly, however, and unlike in adipocytes, immunoisolated GLUT4 vesicles from rat skeletal muscle contained no detectable immunoreactivity towards the transferrin receptor, suggesting that Rab4 was present in a GLUT4 IM pool that was not characteristic of early endosomes. The involvement of Rab4 in GLUT4 translocation was further supported by the finding that insulin treatment resulted in a significant (43%) reduction in Rab4 in the IM compartment. Our results suggest (i) that insulin induces the loss of both GLUT4 and Rab4 from the same IM compartment, (ii) that Rab4 may be involved in GLUT4 translocation based on its coprecipitation with the transporter from the insulin-sensitive pool and (iii) that Rab4 can be localized to intracellular membranes which appear not to be of early endosome origin.