In several cell types, specific membrane proteins are retained intracellularly and rapidly redistributed to the surface in response to stimulation. In fat and muscle, the GLUT4 glucose transporter is dynamically retained because it is rapidly internalized and slowly recycled to the plasma membrane. Insulin increases the recycling of GLUT4, resulting in a net translocation to the surface. We have shown that fibroblasts also have an insulin-regulated recycling mechanism. Here we show that GLUT4 is retained within the transferrin receptor-containing general endosomal recycling compartment in Chinese hamster ovary (CHO) cells rather than being segregated to a specialized, GLUT4-recycling compartment. With the use of total internal reflection microscopy, we demonstrate that the TR and GLUT4 are transported from the pericentriolar recycling compartment in separate vesicles. These data provide the first functional evidence for the formation of distinct classes of vesicles from the recycling compartment. We propose that GLUT4 is dynamically retained within the endosomal recycling compartment in CHO cells because it is concentrated in vesicles that form more slowly than those that transport TR. In 3T3-L1 adipocytes, cells that naturally express GLUT4, we find that GLUT4 is partially segregated to a separate compartment that is inaccessible to the TR. We present a model for the formation of this specialized compartment in fat cells, based on the general mechanism described in CHO cells, which may explain the increased retention of GLUT4 and its insulin-induced translocation in fat cells.