Skeletal muscle surface membrane is constituted by the PM domain and its specialized deep invaginations known as TTs. We have shown previously that insulin induces a rapid translocation of GLUT4s from an IM pool to the PM in rat skeletal muscle (6). In this study, we have investigated the possibility that insulin also stimulates the translocation of GLUT4 proteins to TTs, which constitute the largest area of the cell surface envelope. PM, TTs, and IM components of control and insulinized skeletal muscle were isolated by subcellular fractionation. The TTs then were purified further by removing vesicles of SR origin by using a Ca-loading procedure. Ca-loading resulted in a five- to sevenfold increase in the purification of TTs in the unloaded fraction relative to the loaded fraction, assessed by immunoblotting with an anti-OHP-receptor monoclonal antibody. In contrast, estimation of the content of Ca²⁺-ATPase protein (a marker of SR) with a specific polyclonal antibody revealed that most, if not all, SR vesicles were recovered in the Ca-loaded fraction. Western blotting with an anti-COOH-terminal GLUT4 protein polyclonal antibody revealed that acute insulin injection in vivo (30 min) increased the content of GLUT4 (by 90%) in isolated PMs and markedly enhanced (by 180%) GLUT4 content in purified TTs. Importantly, these insulin-dependent changes in GLUT4 content of PM and purified TTs were seen in the absence of changes in the α₁-subunit of the Na⁺-K⁺-ATPase, a surface membrane marker. Isolated IM components such as LSR, HSR, and triads (terminal cisternae plus junctional TT) contained low or barely detectable amounts of GLUT4; furthermore, insulin treatment did not change the distribution of the transporter protein within these fractions. In contrast, a unique IM fraction that was not associated with either SR or triad markers, contained significant amounts of GLUT4 and showed an insulin-dependent decrease (40%) in GLUT4 protein content. These results show that acute insulin treatment induces the translocation of GLUT4s to both the PM and TTs from a unique intracellular organelle not associated with the SR.