In skeletal muscle, acute insulin treatment results in the recruitment of the GLUT4 glucose transporter from intracellular vesicular structures to the plasma membrane. The precise nature of these intracellular GLUT4 stores has, however, remained poorly defined. Using an established skeletal-muscle fractionation procedure we present evidence for the existence of two distinct intracellular GLUT4 compartments. We have shown that after fractionation of crude muscle membranes on a discontinuous sucrose gradient the majority of the GLUT4 immunoreactivity was largely present in two sucrose fractions (30 and 35%, w/w, sucrose; denoted F30 and F35 respectively) containing intracellular membranes of different buoyant densities. Here we show that these fractions contained 44+/-6 and 49+/-7% of the crude membrane GLUT4 reactivity respectively, and could be further discriminated on the basis of their immunoreactivity against specific subcellular antigen markers. Membranes from the F30 fraction were highly enriched in transferrin receptor (TfR) and annexin II, two markers of the early endosome compartment, whereas they were significantly depleted of both GLUT1 and the alpha1-subunit of (Na++K+)-ATPase, two cell-surface markers. Insulin treatment resulted in a significant reduction in GLUT4 content in membranes from the F35 fraction, whereas the amount of GLUT4 in the less dense (F30) fraction remained unaffected by insulin. Immunoprecipitation of GLUT4-containing vesicles from both intracellular fractions revealed that TfR was present in GLUT4 vesicles isolated from membranes from the F30 fraction. In contrast, GLUT4 vesicles from the F35 fraction were devoid of TfR. The aminopeptidase, vp165, was present in GLUT4 vesicles from both F30 and F35; however, vesicles isolated from F30 contained over twice as much vp165 per unit of GLUT4 than those isolated from F35. The biochemical co-localization of vp165/GLUT4 was further substantiated by double-immunogold labelling of ultrathin muscle sections. Overall, our data indicate the presence of at least two internal GLUT4 pools: one possibly derived from an endosomal recycling compartment, and the other representing a specialized insulin-sensitive GLUT4 storage pool. Both pools contain vp165.