Vesicles containing the glucose transporter GLUT4 from rat adipocytes contain a major protein of 110 kDa. We have isolated this protein, obtained the sequences of peptides, and cloned a large portion of its cDNA. This revealed that the protein is sortilin, a novel membrane protein that was cloned in another context from a human source while this work was in progress. Subcellular fractionation of rat and 3T3-L1 adipocytes, together with GLUT4 vesicle isolation, showed that sortilin was primarily located in the low density microsomes in vesicles containing GLUT4. Insulin caused a 1.7-fold increase in the amount of sortilin at the plasma membranes of 3T3-L1 adipocytes, as assessed by cell surface biotinylation. The expression of sortilin in 3T3-L1 cells occurred only upon differentiation. Previous characterization of sortilin has led to the suggestion that it functions to sort lumenal proteins from the trans Golgi. The significance of its insulin-stimulated increase at the cell surface and of its expression upon differentiation will require definitive delineation of its function.